Transgenic expression cassettes for expressing nucleic acids in non-reproductive floral tissues of plants

ABSTRACT

The invention relates to methods for the specific, transgenic expression of nucleic acid sequences in non-reproductive flower tissues of plants, and expression cassettes and expression vectors containing promoters which have an expression specificity for non-productive tissues of the flower. The invention also relates to organisms (preferably plants), cultures, parts, or reproduction material derived therefrom, which are transformed by means of said transgenic expression cassettes or expression vectors, and the use thereof for producing food, fodder, seeds, pharmaceuticals, or fine chemicals.

The invention relates to methods for the targeted transgenic expressionof nucleic acid sequences in nonreproductive floral tissues of plants,and to transgenic expression cassettes and expression vectors whichcomprise promoters having an expression specificity for nonreproductivefloral tissues of plants. The invention further relates to organisms(preferably plants) transformed with these transgenic expressioncassettes or expression vectors, to cultures, parts or propagationmaterial derived therefrom, and to the use of the same for producinghuman and animal foods, seeds, pharmaceuticals or fine chemicals.

The aim of biotechnological operations on plants is to produce plantswith advantageous novel properties, for example for increasing theagricultural productivity, for increasing the quality of human foods orfor producing particular chemicals or pharmaceuticals (Dunwell J M(2000) J Exp Bot 51 Spec No:487-96). A basic precondition for transgenicexpression of particular genes is the provision of plant-specificpromoters. Promoters are important tools in plant biotechnology forcontrolling the expression of particular genes in a transgenic plant andthus achieving particular traits of the plant.

Various plant-specific promoters are known, for example constitutivepromoters such as the promoter of the Agrobacterium nopaline synthase,the TR dual promoter or the promoter of the cauliflower mosaic virus(CaMV) 35S transcript (Odell et al. (1985) Nature 313:810-812). Adisadvantage of these promoters is that they are constitutively activein virtually all tissues of the plant. Targeted expression of genes inparticular plant parts or at particular times of development is notpossible with these promoters.

Promoters having specificities for various plant tissues such asanthers, ovaries, flowers, leaves, stalks, roots, tubers or seeds havebeen described. The stringency of the specificity and the expressionactivity of these promoters varies widely.

The flower of plants serves for sexual reproduction of flowering plants.The flowers of plants—especially the petals—frequently accumulate largeamounts of secondary plant products such as, for example, terpenes,anthocyans, carotenoids, alkaloids and phenylpropanoids, which serve asscents, defensive substances or as colorants in the flower. Many ofthese substances are of commercial interest. In addition, the flower budand the flower of the plant is a sensitive organ, especially to stressfactors such as cold.

The Arabidopsis thaliana gene locus At3g01980 (GenBank Acc.-No.:NC_(—)003074; Arabidopsis thaliana chromosome 3; base pairs: complement327677 to 329029) encodes a putative dehydrogenase (derived cDNA:GenBank Acc.-No: NM_(—)111064; SEQ ID NO: 11). The Arabidopsis thalianagene locus At1g63140 (GenBank Acc.-No: NC_(—)003070.2; Arabidopsisthaliana chromosome 1; base pairs 23069430 to 23070871) encodes aputative caffeic acid o-methyltransferase (derived cDNA: GenBankAcc.-No: NM_(—)104992.1; SEQ ID NO: 13). The precise function,transcription and the expression patterns of these genes are notdescribed.

Flower-specific promoters such as, for example, the phytoene synthasepromoter (WO 92/16635), the promoter of the P-rr gene (WO 98/22593) orthe promoter of the APETALA3 gene (Hill T A et al. (1998) Development125:1711-1721) are known. However, all these promoters have one or moredisadvantages which are prejudicial to wide use:

-   1) within the flower they are specific for one or more flower    tissues and do not guarantee expression in all tissues of the    flower.-   2) they are—as in the example of the APETALA3 gene which is involved    in flower development—highly regulated during flower development and    are not active in all phases of flower development.-   3) they occasionally show strong secondary activities in other plant    tissues. Thus, the known promoters (such as, for example, the    APETALA3 promotor) show in most cases an activity in seeds, anthers    and the ovaries of the flower, which constitute sensitive floral    organs which are directly involved in the plants' reproduction.    Expression here is in many cases unnecessary and disadvantageous    since it may interfere with the plants' reproduction. Moreover, the    expressed gene product can be dispersed in an undesired manner by    seeds and pollen in the air. For the purposes of a biotechnological    exploitation of transgenic plants, this is largely to be avoided.

Despite the large number of known plant promoters, no promoter with aspecificity for the plant flower which essentially lacks expression inthe pollens and ovaries, i.e. which is only active in thenonreproductive tissues, has been identified to date, nor are anypromoters known which, in addition to having the abovementionedspecificity, are active essentially during all of the floraldevelopment.

It is therefore an object to provide methods and suitable promoters forthe targeted, transgenic expression of nucleic acids into thenonreproductive floral tissues. We have found that this object isachieved by providing the promoters of the genes with the gene locusnames At3g01980 (hereinbelow “76L” promoter; SEQ ID NO: 1) and At1g63140(hereinbelow “84L” promoter; SEQ ID NO: 2) from Arabidopsis thaliana.These promoters show expression in all floral organs with the exceptionof the pollen and the ovaries. This expression pattern can be observedin the flower bud, the flower and the wilting flower.

A first aspect of the invention relates to methods for the targetedtransgenic expression of nucleic acid sequences in nonreproductivefloral tissues of plants, comprising the following steps:

-   I. introduction of a transgenic expression cassette into plant    cells, wherein the transgenic expression cassette comprises at least    the following elements    -   a) at least one promoter sequence selected from the group of        sequences consisting of        -   i) the promoter sequences of SEQ ID NO: 1 or 2 and        -   ii) functional equivalents of the promoter sequences of SEQ            ID NO: 1 or 2 with essentially the same promoter activity as            a promoter of SEQ ID NO: 1 or 2 and        -   iii) functional equivalent fragments of the sequences of i)            or ii) with essentially the same promoter activity as a            promoter of SEQ ID NO: 1 or 2, and    -   b) at least one further nucleic acid sequence, and    -   c) optionally further genetic control elements,    -   wherein at least one promoter sequence and one further nucleic        acid sequence are functionally linked together, and the further        nucleic acid sequence is heterologous in relation to the        promoter sequence, and-   II. selection of transgenic cells which comprise said expression    cassette stably integrated into the genome, and-   III. regeneration of complete plants from said transgenic cells,    wherein at least one of the further nucleic acid sequences is    expressed essentially in all nonreproductive floral tissues, but    essentially not in the pollen and the ovaries.

A further aspect relates to transgenic expression cassettes as, forexample, can be employed in the method of the invention. The transgenicexpression cassettes preferably comprise for the targeted transgenicexpression of nucleic acid sequences in nonreproductive floral tissuesof plants,

-   a) at least one promoter sequence selected from the group of    sequences consisting of    -   i) the promoter sequences of SEQ ID NO: 1 or 2 and    -   ii) functional equivalents of the promoter sequences of SEQ ID        NO: 1 or 2 with essentially the same promoter activity as a        promoter of SEQ ID NO: 1 or 2 and    -   iii) functionally equivalent fragments of the sequences of i)        or ii) with essentially the same promoter activity as a promoter        of SEQ ID NO: 1 or 2, and-   b) at least one further nucleic acid sequence, and-   c) optionally further genetic control elements,    wherein at least one promoter sequence and one further nucleic acid    sequence are functionally linked together, and the further nucleic    acid sequence is heterologous in relation to the promoter sequence.

The expression cassettes of the invention may comprise further geneticcontrol sequences and/or additional functional elements.

It is possible and preferred for the transgenic expression cassettes tomake possible, through the nucleic acid sequence to be expressedtransgenically, the expression of a protein encoded by said nucleic acidsequence and/or the expression of a sense-RNA, antisense-RNA ordouble-stranded RNA encoded by said nucleic acid sequence.

A further aspect of the invention relates to transgenic expressionvectors which comprise one of the expression cassettes of the invention.

A further aspect of the invention relates to transgenic organisms whichcomprise one of the expression cassettes or expression vectors of theinvention. The organism can be selected from the group consisting ofbacteria, yeasts, fungi, nonhuman animal and plant organisms or ofcells, cell cultures, parts, tissues, organs or propagation materialderived therefrom, and the organism is preferably selected from thegroup of agricultural crop plants.

A further aspect of the invention therefore relates to the use saidtransgenic organisms or cells, cell cultures, parts, tissues, organs orpropagation material derived therefrom to produce human and animalfoods, seeds, pharmaceuticals or fine chemicals, where the finechemicals are preferably enzymes, vitamins, amino acids, sugars,saturated or unsaturated fatty acids, natural or synthetic flavorings,aromatizing substances or colorants. The invention further includesmethods for producing said human and animal foods, seeds,pharmaceuticals or fine chemicals employing the trangenic organisms ofthe invention or cells, cell cultures, parts, tissues, organs orpropagation material derived therefrom.

The transgenic expression cassettes of the invention are particularlyadvantageous for the following reasons:

-   a) they impart selective expression in nonreproductive tissues of    the flower bud and the flower of plants and make numerous    applications possible, such as, for example, resistance to stress    factors such as cold or targeted synthesis of secondary plant    products. Expression takes place throughout the period of flower bud    and flower development.-   b) they show no expression in reproductive tissues (such as pollen    or ovaries), whereby interference with the reproduction and    spreading of the transgenic protein by pollen or seeds in the air is    avoided.

The transgenic expression cassettes according to the invention, and thetransgenic expression vectors and transgenic organisms derived fromthem, may comprise functional equivalents of the promoter sequencesdescribed under SEQ ID NO: 1 or 2.

The promoter activity of a functionally equivalent promoter is referredto as being “essentially the same” when the transcription of aparticular nucleic acid sequence to be expressed transgenically underthe control of said functionally equivalent promoter under otherwiseunchanged conditions shows a targeted expression in essentially allnonreproductive floral tissues, with essentially no expression in thepollen and ovaries.

“Flower” generally means a shoot of limited growth whose leaves havebeen transformed into reproductive organs. The flower consists ofvarious “floral tissues” such as, for example, the sepals, the petals,the stamens or the carpels. Androecium is the term used for the totalityof stamens in the flower. The stamens are located within the circle ofpetals and sepals. A stamen is composed of a filament and of an antherlocated at the end. The latter in turn is divided into two thecae whichare connected together by a connective. Each theca consists of twopollen sacs in which the pollen is formed.

“Nonreproductive floral tissue” refers to all tissues of the flowerexcept for the pollen and the ovaries.

“Essentially all nonreproductive floral tissues” means with regard tothe nonreproductive floral tissues that some of these tissues, either intotal or at specific points in time of their development, may display nosubstantial expression, where these tissues however amount to preferablyless than 20% by weight, preferably less than 10% by weight, especiallypreferably less than 5% by weight, very especially preferably less than1% by weight of the total weight of the nonreproductive floral tissues.

“Targeted” means in relation to the expression in nonreproductive floraltissues preferably that the expression under the control of one of thepromoters of the invention in the nonreproductive floral tissues is atleast ten times, particularly preferably at least fifty times, veryparticularly preferably at least one hundred times that of anothertissue such as, for example, the pollen or the ovaries or a nonfloraltissue, such as the leaves.

The fact that the promoters according to the invention “show essentiallyno expression in the pollen and ovaries” preferably means that thestatistical mean of the expression over all reproductive floral tissuesis at most 10%, preferably at most 5%, most preferably at most 1% thatof the statistical mean of the expression over all nonreproductivefloral tissues under identical conditions.

Preferably, the expression within the nonreproductive floral tissues isessentially constant. “Essentially constant” in this context preferablymeans that the standard deviation of the expression between theindividual nonreproductive floral tissues relative to the statisticalmean of the expression over all nonreproductive floral tissues is lessthan 50%, preferably 20%, especially preferably 10%, very especiallypreferably 5%.

Preferably, the expression within at least one particularnonreproductive floral tissue is essentially constant over alldevelopmental stages of the flower. “Essentially constant” in thiscontext preferably means that the standard deviation of the expressionbetween the individual points in time of the development of theparticular nonreproductive floral tissue relative to the statisticalmean of the expression over all points in time of the development isless than 50%, preferably 20%, especially preferably 10%, veryespecially preferably 5%.

The nucleic acid sequences in functional linkage with the test promoterwhich are preferably employed for estimating the level of expression arethose which code for easily quantifiable proteins. Very particularpreference is given in this connection to reporter proteins (SchenbornE, Groskreutz D. (1999) Mol Biotechnol 13(1): 29-44) such as the greenfluorescence protein (GFP) (Chiu W L et al. (1996) Curr Biol 6:325-330;Leffel S M et al. (1997) Biotechniques 23(5):912-8), chloramphenicoltransferase, luciferase (Millar et al. (1992) Plant Mol Biol Rep10:324-414), β-glucuronidase or β-galactosidase. Very particularpreference is given to β-glucuronidase (Jefferson et al. (1987) EMBO J6:3901-3907).

“Conditions which are otherwise unchanged” means that the expressioninitiated by one of the transgenic expression cassettes to be comparedis not modified by combination with additional genetic controlsequences, for example enhancer sequences. Unchanged conditions furthermeans that all general conditions such as, for example, plant species,stage of development of the plants, culture conditions, assay conditions(such as buffer, temperature, substrates etc.) are kept identicalbetween the expressions to be compared.

“Transgenic” means—for example in relation to an expression cassette (orto an expression vector or transgenic organism comprising the former)all those constructions which have originated by recombinant methods inwhich either

-   a) the promoter of SEQ ID NO: 1 or 2 or a functional equivalent    thereof or a functional equivalent part of the above, or-   b) a further nucleic acid sequence which is functionally linked with    a), or-   c) (a) and (b)    are not in their natural genetic environment or have been modified    by recombinant methods, it being possible for the modification to    be, for example, a substitution, addition, deletion, inversion or    insertion of one or more nucleotide residues. The promoter sequence    of the invention (e.g. the sequence as shown in SEQ ID NO: 1, 2, 3    or 4) comprised in the expression cassettes is preferably    heterologous in relation to the further nucleic acid sequence which    is to be expressed transgenically and is functionally linked    thereto. “Heterologous” means in this connection that the further    nucleic acid sequence does not code for the gene which is naturally    under the control of said promoter.

“Natural genetic environment” means the natural chromosomal locus in theoriginal organism or the presence in a genomic library. In the case of agenomic library, the natural, genetic environment of the nucleic acidsequence is preferably still retained at least in part. The environmentflanks the nucleic acid sequence at least on one side and has a sequencelength of at least 50 bp, preferably at least 500 bp, particularlypreferably at least 1000 bp, very particularly preferably at least 5000bp. A naturally occurring expression cassette—for example the naturallyoccurring combination of the promoter of a gene coding for a protein asshown in SEQ ID NO: 12 or 14 or a functional equivalent thereof with itscorresponding coding sequences becomes a trangenic expression constructwhen the latter is modified by unnatural, synthetic (“artificial”)methods such as, for example, a mutagenesis. Appropriate methods aredescribed (U.S. Pat. No. 5,565,350; WO 00/15815; see also above).

“Transgenic” means in relation to an expression (“transgenicexpression”) preferably all expressions caused by use of a transgenicexpression cassette, transgenic expression vector or transgenicorganism—complying with the definitions given above.

Functional equivalents of a promoter of SEQ ID NO: 1 or 2 in particularmeans natural or artificial mutations of a promoter of SEQ ID NO: 1 or 2and homologous sequences from other organisms, preferably from plantorganisms, which have essentially the same promoter activity as one ofthe promoters of SEQ ID NO: 1 or 2.

Functional equivalents also comprises all those sequences which arederived from the complementary counterstrand of the sequences defined bySEQ ID NO: 1 or 2 and have essentially the same promoter activity.

Functional equivalents to the promoters of SEQ ID NO: 1 or 2 preferablycomprise those sequences which

-   a) have essentially the same promoter activity as one of the    promoters of SEQ ID NO: 1 or 2 and-   b) have a homology of at least 50%, preferably 70%, more preferably    at least 80%, particularly preferably at least 90%, very    particularly preferably at least 95%, most preferably 99%, with the    sequence of one of the promoters of SEQ ID NO: 1 or 2, wherein the    homology extends over a length of at least 100 base pairs,    preferably at least 200 base pairs, particularly preferably of at    least 300 base pairs, very particularly preferably of at least 400    base pairs, most preferably of at least 500 base pairs.

It is possible in this connection for the level of expression of thefunctional equivalents to differ both downwards and upwards from acomparison value. Preference is given in this connection to thesequences whose level of expression, measured on the basis of thetranscribed mRNA or the subsequently translated protein, underconditions which are otherwise unchanged differs quantitatively by notmore than 50%, preferably 25%, particularly preferably 10%, from acomparison value obtained with the promoters described by SEQ ID NO: 1or 2. Particularly preferred sequences are those whose level ofexpression, measured on the basis of the transcribed mRNA or thesubsequently translated protein, under conditions which are otherwiseunchanged exceeds quantitatively by more than 50%, preferably 100%,particularly preferably 500%, very particularly preferably 1000%, acomparison value obtained with the promoter described by SEQ ID NO: 1 or2.

Examples of promoter sequences employed in the transgenic expressioncassettes or transgenic expression vectors of the invention can easilybe found for example in various organisms whose genomic sequence isknown, such as, for example, Arabidopsis thaliana, Brassica napus,Nicotiana tabacum, Solanum tuberosum, Helianthium annuus, Linum sativum,by making homology comparisons in databases. A possible and preferredstarting point for this is the coding regions of the genes whosepromoters are described by SEQ ID NO: 1 or 2. Starting from, forexample, the cDNA sequences of these genes described by SEQ ID NO: 11 or13 or the protein sequences derived therefrom and described by SEQ IDNO: 12 or 14 it is possible easily to identify, in a manner familiar tothe skilled worker, the corresponding homologous genes in other plantspecies by screening databases or gene libraries (using appropriate geneprobes).

In a preferred embodiment of the invention, functional equivalents ofthe promoter described by SEQ ID NO: 1 comprise all those promoterswhich are located in a plant organism in the 5′ direction upstream of agenomic sequence which encodes a protein with at least 60%, preferablyat least 80%, especially preferably at least 90%, most preferably atleast 95% homology with the protein of SEQ ID NO: 12, wherein saidpromoters constitute the natural promoter of said genomic sequence.Especially preferably, functional equivalents of the promoter describedby SEQ ID NO: 1 comprise all those promoters which are located in aplant organism in the 5′ direction upstream of a genomic sequence whichencodes a nucleic acid sequence whose derived cDNA has at least 60%,preferably at least 80%, especially preferably at least 90%, mostpreferably at least 95% homology with the nucleic acid sequence as shownin SEQ ID NO: 11, wherein said promoters constitute the natural promoterof said genomic sequence. Preferred promoters comprise a sequence regionof least 250 base pairs, preferably at least 500 base pairs,particularly preferably 1000 base pairs, most preferably at least 2000base pairs, in the 5′ direction calculated from the ATG start codon ofsaid genomic sequences. Functional equivalents of the promoter describedby SEQ ID NO: 1 are particularly preferably all promoters which arelocated in a plant organism in the 5′ direction upstream of a genomicsequence which encodes a protein which comprises at least one of thefollowing sequence motifs: 1. NGD(E/Q)VSRNIA (SEQ ID NO: 23) 2.LAKHGC(R/K)LV (SEQ ID NO: 24) 3. MGNEXSLRSXVDXIR (SEQ ID NO: 25) 4.TYQGKXQDILXVS(Q/E)DEF (SEQ ID NO: 26) 5. IT(K/R)INLTAXWFXLKAVA (SEQ IDNO: 27)

Very particularly preferred functional equivalents of the promoterdescribed by SEQ ID NO: 1 are those promoters which are located in aplant organism in the 5′ direction upstream of a genomic sequence whichencodes a protein, wherein said protein comprises at least one of thefollowing sequences:

-   1. the homologous sequence (H2) from oilseed rape as shown in SEQ ID    NO: 16-   2. the homologous sequence (H3) from oilseed rape as shown in SEQ ID    NO: 18

Most preferred functional equivalents of the promoter described by SEQID NO: 1 are those promoters which are located in a plant organism inthe 5′ direction upstream of a genomic sequence which encodes a nucleicacid sequence whose derived cDNA comprises at least one of the followingsequences:

-   1. the homologous sequence (H2) from oilseed rape as shown in SEQ ID    NO: 15-   2. the homologous sequence (H3) from oilseed rape as shown in SEQ ID    NO: 17

In a preferred embodiment of the invention, functional equivalents ofthe promoter described by SEQ ID NO: 2 comprise all those promoterswhich are located in a plant organism in the 5′ direction upstream of agenomic sequence which encodes a protein with at least 60%, preferablyat least 80%, especially preferably at least 90%, most preferably atleast 95% homology with the protein of SEQ ID NO: 14, wherein saidpromoters constitute the natural promoter of said genomic sequence.Especially preferably, functional equivalents of the promoter describedby SEQ ID NO: 2 comprise all those promoters which are located in aplant organism in the 5′ direction upstream of a genomic sequence whichencodes a nucleic acid sequence whose derived cDNA has at least 60%,preferably at least 80%, especially preferably at least 90%, mostpreferably at least 95% homology with the nucleic acid sequence as shownin SEQ ID NO: 13, wherein said promoters constitute the natural promoterof said genomic sequence. Preferred promoters comprise a sequence regionof least 250 base pairs, preferably at least 500 base pairs,particularly preferably 1000 base pairs, most preferably at least 2000base pairs, in the 5′ direction calculated from the ATG start codon ofsaid genomic sequences. Functional equivalents of the promoter describedby SEQ ID NO: 2 are particularly preferably all promoters which arelocated in a plant organism in the 5′ direction upstream of a genomicsequence which encodes a protein which comprises at least one of thefollowing sequence motifs: 1. AEPVCTXFL (SEQ ID NO: 28) 2.EGKDXFXSAHGMXXFE (SEQ ID NO: 29) 3. EQFAXMFNXAM (SEQ ID NO: 30) 4.ATXIMKK(V/I)LEVY(K/R)GFED (SEQ ID NO: 31) 5. TLVD(V/I)GGGXGT (SEQ ID NO:32)

Very particularly preferred functional equivalents of the promoterdescribed by SEQ ID NO: 2 are those promoters which are located in aplant organism in the 5′ direction upstream of a genomic sequence whichencodes a protein, wherein said protein comprises at least one of thefollowing sequences:

-   1. the homologous sequence (H4) from oilseed rape as shown in SEQ ID    NO: 20-   2. the homologous sequence (H5) from oilseed rape as shown in SEQ ID    NO: 22

Most preferred functional equivalents of the promoter described by SEQID NO: 2 are those promoters which are located in a plant organism inthe 5′ direction upstream of a genomic sequence which encodes a nucleicacid sequence whose derived cDNA comprises at least one of the followingsequences:

-   1. the homologous sequence (H4) from oilseed rape as shown in SEQ ID    NO: 19-   2. the homologous sequence (H5) from oilseed rape as shown in SEQ ID    NO: 21

A further subject of the invention therefore relates to polypeptidescomprising an amino acid sequence as shown in SEQ ID NO: 16, 18, 20 or22 and the nucleic acid sequences encoding them, preferably thesequences comprising a sequence as shown in SEQ ID NO: 15, 17, 19 or 21or the sequences derived therefrom the result of degeneracy of thegenetic code.

A further aspect of the invention relates to the use of at least onenucleic acid sequence or of a part thereof in methods for identifyingand/or isolating promoters of genes which code for said nucleic acidsequence, wherein said nucleic acid sequence encodes an amino acidsequence comprising at least one sequence as shown in SEQ ID NO: 23, 24,25, 26, 27, 28, 29, 30, 31 or 32 or a variation indicated for thesesequences. Said nucleic acid sequence preferably codes for an amino acidsequence comprising a sequence as shown in SEQ ID NO: 12, 14, 16, 18, 20or 22. Said nucleic acid sequence particularly preferably comprises asequence as shown in SEQ ID NO: 11, 13, 15, 17, 19 or 21. “Part” meansin relation to the nucleic acid sequence preferably a sequence of atleast 10 bases, preferably 15 bases, particularly preferably 20 bases,most preferably 30 bases.

Further included according to the invention are methods for identifyingand/or isolating promoters of genes which encode a promoter havingspecificity for nonreproductive floral tissue, wherein at least onenucleic acid sequence or a part thereof is employed in theidentification and/or isolation, wherein said nucleic acid sequenceencodes an amino acid sequence which comprises at least one sequence asshown in SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 or avariation indicated for these sequences. Said nucleic acid sequencepreferably codes for an amino acid sequence comprising a sequence asshown in SEQ ID NO: 12, 14, 16, 18, 20 or 22. Said nucleic acid sequenceparticularly preferably comprises a sequence as shown in SEQ ID NO: 11,13, 15, 17, 19 or 21. “Part means in relation to the nucleic acidsequence preferably a sequence of at least 10 bases, preferably 15bases, particularly preferably 20 bases, most preferably 30 bases. In apreferred embodiment, the method of the invention is based on thepolymerase chain reaction, wherein said nucleic acid sequence or a partthereof is employed as primer.

Various methods for identifying and isolating the promoter of thecorresponding gene, starting from a nucleic acid sequence (e.g. a genetranscript such as, for example, a cDNA) are known to the skilledworker. In principle, all methods for amplifying flanking chromosomalsequences are available for example for this purpose. The two mostcommonly used methods are inverse PCR (“iPCR”; diagrammatically depictedin FIG. 10) and “Thermal Asymmetric Interlaced PCR” (“TAIL PCR”).

For the iPCR, genomic DNA of the organism from which the functionallyequivalent promoter is to be isolated is completely digested with agiven restriction enzyme, and then the individual fragments arereligated, i.e. linked to themselves to give a circular molecule, in adiluted mixture. The large number of resulting circular DNA moleculesalso includes those comprising the known sequence (for example thesequence coding for the homologous protein). Starting from this, thecircular molecule can be amplified by PCR using a primer pair where bothprimers are able to anneal to the known sequence segment. One possibleembodiment of the iPCR is reproduced in example 6.

The TAIL-PCR is based on the use of firstly a set of successivelytruncated highly specific primers which anneal to the known genomicsequence (for example the sequence coding for the homologous protein),and secondly a set of shorter random primers with a lower meltingtemperature, so that a less sequence-specific annealing to genomic DNAflanking the known genomic sequence takes place. Annealing of theprimers to the DNA to be amplified is possible with such a primercombination making specific amplification of the desired target sequencepossible. One possible embodiment of the TAIL-PCR is reproduced forexample in example 5.

A further aspect of the invention relates to methods for producing atransgenic expression cassette having specificity for nonreproductivefloral tissue, comprising the following steps:

-   I. isolation of a promoter sequence with specificity for    nonreproductive floral tissues, wherein at least one nucleic acid    sequence or a part thereof is employed in the isolation, wherein    said nucleic acid sequence encodes an amino acid sequence which    comprises at least one sequence as shown in SEQ ID NO: 23, 24, 25,    26, 27, 28, 29, 30, 31 or 32 or a variation indicated for these    sequences.-   II. functional linkage of said promoter with a further nucleic acid    sequence, wherein said nucleic acid sequence is heterologous in    relation to the promoter.

Said nucleic acid sequence preferably codes for an amino acid sequencecomprising a sequence as shown in SEQ ID NO: 12, 14, 16, 18, 20 or 22.Said nucleic acid sequence particularly preferably comprises a sequenceas shown in SEQ ID NO: 11, 13, 15, 17, 19 or 21. “Part” means inrelation to the nucleic acid sequence preferably a sequence of at least10 bases, preferably 15 bases, particularly preferably 20 bases, mostpreferably 30 bases. In a preferred embodiment, the method of theinvention is based on the polymerase chain reaction, wherein saidnucleic acid sequence or a part thereof is employed as primer. Methodsknown to the skilled worker, such as, for example, ligation etc., can beemployed for the functional linkage (see below).

-   “Mutation” means substitution, addition, deletion, inversion or    insertion of one or more nucleotide residues. Thus, for example, the    present invention also comprises nucleotide sequences obtained by    modification of the promoters as shown in SEQ ID NO: 1 or 2. The aim    of such a modification may be further localization of the sequence    comprised therein or, for example, also the insertion of further    restriction enzyme cleavage sites, the deletion of excess DNA or the    addition of further sequences, for example further regulatory    sequences.

Where insertions, deletions or substitutions, such as, for example,transitions and transversions, are appropriate, it is possible to usetechniques known per se, such as in vitro mutagenesis, primer repair,restriction or ligation. Transition means a base-pair exchange of apurine/pyrimidine pair into another purine/pyrimidine pair (e.g. A-T forG-C). Transversion means a base-pair exchange of a purine/pyrimidinepair for a pyrimidine/purine pair (e.g. A-T for T-A). Deletion meansremoval of one or more base pairs. Insertion means introduction of oneor more base pairs.

-   Complementary ends of the fragments for ligation can be made    available by manipulations such as, for example, restriction,    chewing back or filling in of overhangs for blunt ends. Analogous    results are also obtainable by using the polymerase chain reaction    (PCR) using specific oligonucleotide primers.

Homology between two nucleic acids means the identity of the nucleicacid sequence over the complete sequence length in each case, which iscalculated by comparison with the aid of the GAP program algorithm(Wisconsin Package Version 10.0, University of Wisconsin, GeneticsComputer Group (GCG), Madison, USA), setting the following parameters:

-   -   Gap Weight: 12 Length Weight: 4    -   Average Match: 2.912 Average Mismatch:-2.003

For example, a sequence which has a homology of at least 50% based onnuleic acids with the sequence SEQ ID NO: 1 means a sequence which has ahomology of at least 50% on comparison with the sequence SEQ ID NO: 1 bythe above program algorithm with the above set of parameters.

Homology between two polypeptides means the identity of the amino acidsequence over the respective sequence length, which is calculated bycomparison with the aid of the GAP program algorithm (Wisconsin PackageVersion 10.0, University of Wisconsin, Genetics Computer Group (GCG),Madison, USA), setting the following parameters:

-   -   Gap Weight: 8 Length Weight: 2    -   Average Match: 2.912 Average Mismatch:-2.003

For example, a sequence having a homology of at least 60% based onprotein with the sequence SEQ ID NO: 12 means a sequence which has ahomology of at least 60% on comparison with the sequence SEQ ID NO: 12by the above program algorithm with the above set of parameters.

Functional equivalents also means DNA sequences which hybridize understandard conditions with one of the nucleic acid sequences coding forone of the promoters as shown in SEQ ID NO: 1 or 2 or with the nucleicacid sequences complementary thereto, and which have substantially thesame promoter properties.

The term standard hybridization conditions is to be understood broadlyand means both stringent and less stringent hybridization conditions.Such hybridization conditions are described inter alia in Sambrook J,Fritsch E F, Maniatis T et al., in Molecular Cloning—A LaboratoryManual, 2^(nd) edition, Cold Spring Harbor Laboratory Press, 1989, pages9.31-9.57 or in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the conditions during thewashing step can be selected from the range of conditions limited bythose of low stringency (with approximately 2×SSC at 50° C.) and thoseof high stringency (with approximately 0.2×SSC at 50° C., preferably at65° C.) (20×SSC: 0.3 M sodium citrate, 3 M NaCl, pH 7.0). In addition,the temperature during the washing step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tomore stringent conditions at approximately 65° C. Both parameters, thesalt concentration and the temperature, can be varied simultaneously,and it is also possible for one of the two parameters to be keptconstant and only the other to be varied. It is also possible to employdenaturing agents such as, for example, formamide or SDS during thehybridization. Hybridization in the presence of 50% formamide ispreferably carried out at 42° C. Some exemplary conditions forhybridization and washing step are given below:

(1) Hybridization conditions with for example

-   -   a) 4×SSC at 65° C., or    -   b) 6×SSC, 0.5% SDS, 100 μg/ml denatured fragmented salmon sperm        DNA at 65° C., or    -   c) 4×SSC, 50% formamide, at 42° C., or    -   d) 2× or 4×SSC at 50° C. (low-stringency condition), or    -   e) 2× or 4×SSC, 30 to 40% formamide at 42° C. (low-stringency        condition), or    -   f) 6×SSC at 45° C., or,    -   g) 0.05 M sodium phosphate buffer pH 7.0, 2 mM EDTA, 1% BSA and        7% SDS.        (2) Washing steps with for example    -   a) 0.1×SSC at 65° C., or    -   b) 0.1×SSC, 0.5% SDS at 68° C., or    -   c) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C., or    -   d) 0.2×SSC, 0.1% SDS at 42° C., or    -   e) 2×SSC at 65° C. (low-stringency condition), or    -   f) 40 mM sodium phosphate buffer pH 7.0, 1% SDS, 2 mM EDTA.

Methods for preparing functional equivalents of the invention preferablycomprise the introduction of mutations into one of the promoters asshown in SEQ ID NO: 1 or 2. Mutagenesis may be random, in which case themutagenized sequences are subsequently screened for their properties bya trial and error procedure. Particularly advantageous selectioncriteria comprise for example the level of the resulting expression ofthe introduced nucleic acid sequence in a nonreproductive floral tissue.

Methods for mutagenesis of nucleic acid sequences are known to theskilled worker and include by way of example the use of oligonucleotideswith one or more mutations compared with the region to be mutated (e.g.in a site-specific mutagenesis). Primers with approximately 15 toapproximately 75 nucleotides or more are typically employed, withpreferably about 10 to about 25 or more nucleotide residues beinglocated on both sides of the sequence to be localized. Details andprocedure for said mutagenesis methods are familiar to the skilledworker (Kunkel et al. (1987) Methods Enzymol 154:367-382; Tomic et al.(1990) Nucl Acids Res 12:1656; Upender et al. (1995) Biotechniques18(1):29-30; U.S. Pat. No. 4,237,224). A mutagenesis can also beachieved by treating for example transgenic expression vectorscomprising one of the nucleic acid sequences of the invention withmutagenizing agents such as hydroxylamine.

An alternative possibility is to delete nonessential sequences of apromoter of the invention without significantly impairing the essentialproperties mentioned. Such deletion variants represent functionallyequivalent fragments to the promoters described by SEQ ID NO: 1 or 2 orto functional equivalents thereof. Localization of the promoter sequenceto particular essential regulatory regions can be carried out forexample with the aid of a search routine to search for promoterelements. Particular promoter elements are often present in increasednumbers in the regions relevant for promoter activity. This analysis canbe carried out for example with computer programs such as the PLACEprogram (“Plant Cis-acting Regulatory DNA Elements”; Higo K et al.(1999) Nucl Acids Res 27(1): 297-300), the BIOBASE database Transfac”(Biologische Datenbanken GmbH, Braunschweig; Wingender E et al. (2001)Nucleic Acids Res 29(1):281-3) or the PlantCARE database (Lescot M etal. (2002) Nucleic Acids Res 30(1):325-7).

The functionally equivalent fragments of one of the promoters of theinvention—for example of the promoters described by SEQ ID NO: 1 or2—preferably comprise at least 200 base pair, very particularlypreferably at least 500 base pairs, most preferably at least 1000 basepairs of the 3′ end of the respective promoter of the invention—forexample of the promoters described by SEQ ID NO: 1 or 2—the length beingcalculated from the translation start (“ATG” codon) upstream in the 5′direction. Very especially preferred functionally equivalent fragmentsare the promoter sequences described by SEQ ID NO: 3 or 4. Furtherfunctionally equivalent fragments may be generated for example bydeleting any 5′-untranslated regions still present. For this purpose,the start of transcription of the corresponding genes can be determinedby methods familiar to the skilled worker (such as, for example,5′-RACE), and the 5′-untranslated regions can be deleted by PCR-mediatedmethods or endonuclease digestion.

In transgenic expression cassettes of the invention, at least one of thepromoters of the invention (e.g. described by SEQ ID NO: 1, 2, 3 or 4)is functionally linked to at least one nucleic acid sequence to beexpressed transgenically.

A functional linkage means, for example, the sequential arrangement ofone of the promoters of the invention (e.g. described by SEQ ID NO: 1,2, 3 or 4) with a nucleic acid sequence to be expressed transgenicallyand, where appropriate, further genetic control sequences such as, forexample, a terminator or a polyadenylation sequence in such a way thatthe promoter is able to fulfill its function in the transgenicexpression of the nucleic acid sequence under suitable conditions, andexpression of the nucleic acid sequence (i.e. transcription and, whereappropriate, translation) takes place. “Suitable conditions” means inthis connection preferably the presence of the expression cassette in aplant cell, preferably a plant cell comprised in a nonreproductivefloral tissue of a plant.

Arrangements in which the nucleic acid sequence to be expressedtransgenically is positioned behind one of the promoters of theinvention (e.g. described by SEQ ID NO: 1, 2, 3 or 4), so that the twosequences are covalently connected together, are preferred. In thisconnection, the distance between the promoter sequence and the nucleicacid sequence to be expressed transgenically is preferably less than 200base pairs, particularly preferably less than 100 base pairs, veryparticularly preferably less than 50 base pairs.

Production of a functional linkage and production of a transgenicexpression construct can be achieved by means of conventionalrecombination and cloning techniques as described for example inManiatis T, Fritsch E F and Sambrook J (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor(NY), in Silhavy T J, Berman M L and Enquist L W (1984) Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor (NY) andin Ausubel F M et al. (1987) Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley Interscience. However, furthersequences which have for example the function of a linker withparticular restriction enzyme cleavage sites or of a signal peptide mayalso be positioned between the two sequences. Insertion of sequences mayalso lead to expression of fusion proteins. It is possible and preferredfor the transgenic expression construct, consisting of a linkage ofpromoter and nucleic acid sequence to be expressed, to be integratedinto a vector and be inserted into a plant genome for example bytransformation.

However, an expression cassette also means constructions in which one ofthe promoters of the invention (e.g. described by SEQ ID NO: 1, 2, 3 or4) was, without necessarily having been functionally linked beforehandto a nucleic acid sequence to be expressed, introduced into a hostgenome, for example by targeted homologous recombination or randominsertion, there undertakes regulatory control over endogenous nucleicacid sequences then functionally linked thereto, and controls thetransgenic expression thereof. Insertion of the promoter—for example bya homologous recombination—in front of a nucleic acid coding for aparticular polypeptide results in an expression cassette of theinvention which controls the expression of the particular polypeptideselectively in the nonreproductive floral tissues. It is also possiblefor example for the natural promoter of an endogenous gene to bereplaced by one of the promoters of the invention (e.g. described by SEQID NO: 1, 2, 3 or 4), and for the expression behavior of the endogenousgene to be modified.

A further possibility is also for the promoter to be inserted in such away that antisense RNA to the nucleic acid coding for a particularpolypeptide is expressed. In this way, expression of the particularpolypeptide in the nonreproductive organs of the flower is selectivelydownregulated or switched off.

It is also possible analogously for a nucleic acid sequence which is tobe expressed transgenically to be placed—for example by homologousrecombination—behind the sequence which codes for one of the promotersof the invention (e.g. described by SEQ ID NO: 1, 2, 3 or 4), and whichis located in its natural chromosomal context, so as to result in anexpression cassette of the invention which controls the expression ofthe nucleic acid sequence to be expressed transgenically in thenonreproductive floral tissues.

The transgenic expression cassettes of the invention may comprisefurther genetic control sequences. The term genetic control sequences isto be understood broadly and means all sequences having an influence onthe coming into existence or the function of a transgenic expressioncassette of the invention. Genetic control sequences modify for examplethe transcription and translation in prokaryotic or eukaryoticorganisms. The transgenic expression cassettes of the inventionpreferably comprise as additional genetic control sequence a terminatorsequence 3′ downstream from the particular nucleic acid sequence to beexpressed transgenically, and where appropriate further customaryregulatory elements, in each case functionally linked to the nucleicacid sequence to be expressed transgenically.

Genetic control sequences also comprise further promoters, promoterelements or minimal promoters able to modify the expression-controllingproperties. It is thus possible for example through genetic controlsequences for tissue-specific expression to take place additionally independence on particular stress factors. Corresponding elements aredescribed for example for water stress, abscisic acid (Lam E and Chua NH, J Biol Chem 1991; 266(26): 17131-17135) and heat stress (Schoffl F etal. (1989) Mol Gen Genetics 217(2-3): 246-53).

A further possibility is for further promoters which make transgenicexpression possible in further plant tissues or in other organisms suchas, for example, E. coli bacteria to be functionally linked to thenucleic acid sequence to be expressed. Suitable promoters are inprinciple all plant-specific promoters. Plant-specific promoters meansin principle every promoter able to control the expression of genes, inparticular foreign genes, in plants or plant parts, cells, tissues,cultures. It is moreover possible for expression to be for exampleconstitutive, inducible or development-dependent. Preference is given toconstitutive promoters, tissue-specific promoters, development-dependentpromoters, chemically inducible, stress-inducible or pathogen-induciblepromoters. Corresponding promoters are generally known to the skilledworker.

Further advantageous control sequences are to be found for example inthe promoters of gram-positive bacteria such as amy and SPO2 or in theyeast or fungal promoters ADC1, MFa, AC, P-60, CYC1, GAPDH, TEF, rp28,ADH.

It is possible in principle for all natural promoters with theirregulatory sequences like those mentioned above to be used for themethod of the invention. It is additionally also possible for syntheticpromoters to be used advantageously.

Genetic control sequences further comprise also the 5′-untranslatedregions, introns or noncoding 3′-region of genes such as, for example,the actin-1 intron, or the Adh1-S introns 1, 2 and 6 (generally: TheMaize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y.(1994)), preferably the genes with the gene locus At3g01980 andAt1g63140 from Arabidopsis thaliana. It is possible to show that suchregions may have a significant function in regulating gene expression.Thus, it has been shown that 5′-untranslated sequences are able toenhance the transient expression of heterologous genes. Examples oftranslation enhancers which may be mentioned are the 5′ leader sequencefrom the tobacco mosaic virus (Gallie et al. (1987) Nucl Acids Res15:8693-8711) and the like. They may in addition promote tissuespecificity (Rouster J et al. (1998) Plant J 15:435-440). The nucleicacid sequences indicated in SEQ ID NO: 1, 2, 3 or 4 in each caserepresent the promoter region and the 5′-untranslated regions up tobefore the ATG start codon of the respective genes with the gene locusAt3g01980 and At1g63140.

The transgenic expression construct may advantageously comprise one ormore so-called enhancer sequences functionally linked to the promoter,which make increased transgenic expression of the nucleic acid sequencepossible. Additional advantageous sequences can also be inserted at the3′ end of the nucleic acid sequences to be expressed transgenically,such as further regulatory elements or terminators. The nucleic acidsequences to be expressed transgenically may be comprised in one or morecopies in the gene construct.

Polyadenylation signals suitable as control sequences are plantpolyadenylation signals, preferably those which are essentially T-DNApolyadenylation signals from Agrobacterium tumefaciens. Examples ofparticularly suitable terminator sequences are the OCS (octopinesynthase) terminator and the NOS (nopaline synthase) terminator.

Control sequences additionally mean those which make homologousrecombination or insertion into the genome of a host organism possibleor allow deletion from the genome. In homologous recombination forexample the coding sequence of a particular endogenous gene can bespecifically replaced by a sequence coding for a dsRNA. Methods such ascre/lox technology permit tissue-specific, and in some circumstancesinducible, deletion of the transgenic expression construct from thegenome of the host organism (Sauer B (1998) Methods 14(4):381-92). Inthis case, particular flanking sequences are attached to the target gene(lox sequences) and make later deletion by means of cre recombinasepossible.

A transgenic expression cassette and/or the transgenic expressionvectors derived therefrom may comprise further functional elements. Theterm functional element is to be understood broadly and means allelements which have an influence on the production, replication orfunction of the transgenic expression constructs of the invention, ofthe transgenic expression vectors or of the transgenic organisms.Non-restrictive examples which may be mentioned are:

-   a) Selection markers which confer resistance to biocides such as    metabolism inhibitors (e.g. 2-deoxyglucose 6-phosphate;

WO 98/45456), antibiotics (e.g. kanamycin, G 418, bleomycin, hygromycin)or—preferably—herbicides (e.g. phosphinothricin). Examples of selectionmarkers which may be mentioned are: phosphinothricin acetyltransferases(bar and pat gene), which inactivate glutamine synthase inhibitors,5-enolpyruvylshikimate-3-phosphate synthases (EPSP synthase genes) whichconfer resistance to glyphosate (N-(phosphonomethyl)glycine),glyphosate-degrading enzymes (gox gene product; glyphosateoxidoreductase), dehalogenases which for example inactivate dalapon (dehgene product), sulfonylurea- and imidazolinone-inactivating acetolactatesynthases, and nitrilases which for example degrade bromoxynil (bxn geneproduct), the aasa gene product which confers resistance to theantibiotic apectinomycin, streptomycin phosphotransferases (SPT) whichensure resistance to streptomycin, neomycin phosphotransferases (NPTII)which confer resistance to kanamycin or geneticidin, the hygromycinphosphotransferases (HPT) which mediate resistance to hygromycin, theacetolactate synthases (ALS) which confer resistance to sulfonylureaherbicides (e.g. mutated ALS variants with, for example, the S4 and/orHra mutation).

-   b) Reporter genes which code for easily quantifiable proteins and    ensure via an intrinsic color or enzymic activity an assessment of    the transformation efficiency or of the location or timing of the    expression. Very particular preference is given in this connection    to reporter proteins (Schenborn E, Groskreutz D. Mol Biotechnol.    1999; 13(1): 29-44) such as the green fluorescence protein (GFP)    (Sheen et al. (1995) Plant Journal 8(5):777-784), the    chloramphenicol transferase, a luciferase (Ow et al. (1986) Science    234:856-859), the aequorin gene (Prasher et al. (1985) Biochem    Biophys Res Commun 126(3):1259-1268), the β-galactosidase, with very    particular preference for β-glucuronidase (Jefferson et al. (1987)    EMBO J 6:3901-3907).-   c) Origins of replication which ensure replication of the transgenic    expression constructs or transgenic expression vectors of the    invention in, for example, E. coli. Examples which may be mentioned    are ORI (origin of DNA replication), the pBR322 ori or the P15A ori    (Sambrook et al.: Molecular Cloning. A Laboratory Manual, 2^(nd) ed.    Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,    1989).-   d) Elements which are necessary for agrobacterium-mediated plant    transformation, such as, for example, the right or left border of    the T-DNA or the vir region.

“Introduction” comprises for the purposes of the invention all methodssuitable for introducing a nucleic acid sequence (for example anexpression cassette of the invention) directly or indirectly into anorganism (e.g. a plant) or a cell, compartment, tissue, organ orpropagation material (e.g. seeds or fruits) thereof, or for generatingsuch therein. Direct and indirect methods are comprised. Theintroduction can lead to a temporary (transient) presence of saidnucleic acid sequence or else to a permanent (stable) presence.Introduction comprises for example methods such as transfection,transduction or transformation. The organisms used in the methods aregrown or cultured, depending on the host organism, in the manner knownto the skilled worker.

Introduction of a transgenic expression cassette of the invention intoan organism or cells, tissues, organs, parts or seeds thereof(preferably into plants or plant cells, tissues, organs, parts or seeds)can advantageously be achieved by use of vectors comprising thetransgenic expression cassettes. Vectors may be for example plasmids,cosmids, phages, viruses or else agrobacteria. The transgenic expressioncassettes can be inserted into the vector (preferably a plasmid vector)via a suitable restriction cleavage site. The resulting vector can befirstly introduced and amplified in E. coli. Correctly transformed E.coli are selected and cultured, and the recombinant vector is isolatedby methods familiar to the skilled worker. Restriction analysis andsequencing can be used to check the cloning step.

Preferred vectors are those making stable integration of the expressioncassette into the host genome possible. Production of a transformedorganism (or of a transformed cell or tissue) requires introduction ofthe appropriate DNA (e.g. the expression vector) or RNA into theappropriate host cell. A large number of methods is available for thisprocess, which is referred to as transformation (or transduction ortransfection) (Keown et al. (1990) Methods in Enzymology 185:527-537).Thus, the DNA or RNA can for example be introduced directly bymicroinjection or by bombardment with DNA-coated microparticles. Thecell can also be permeabilized chemically, for example with polyethyleneglycol, so that the DNA is able to enter the cell by diffusion. The DNAintroduction can also take place by protoplast fusion with otherDNA-comprising units such as minicells, cells, lysosomes or liposomes.Electroporation is another suitable method for introducing DNA, in whichthe cells are reversibly permeabilized by an electrical impulse.Corresponding methods are described (for example in Bilang et al. (1991)Gene 100:247-250; Scheid et al. (1991) Mol Gen Genet 228:104-112;Guerche et al. (1987) Plant Science 52:111-116; Neuhause et al. (1987)Theor Appl Genet 75:30-36; Klein et al. (1987) Nature 327:70-73; Howellet al. (1980) Science 208:1265; Horsch et al. (1985) Science227:1229-1231; DeBlock et al. (1989) Plant Physiology 91:694-701;Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.)Academic Press Inc. (1988); and Methods in Plant Molecular Biology(Schuler and Zielinski, eds.) Academic Press Inc. (1989)).

Vectors preferred for expression in E. coli are pQE70, pQE60 and pQE-9(QIAGEN, Inc.); pBluescript vectors, Phagescript vectors, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene Cloning Systems, Inc.); ptrc99a, pKK223-3,pKK233-3, pDR540, pRIT5 (Pharmacia Biotech, Inc.).

Preferred vectors for expression in mammalian cells comprise pWLNE0,pSV2CAT, pOG44, pXT1 and pSG (Stratagene Inc.); pSVK3, pBPV, pMSG andpSVL (Pharmacia Biotech, Inc.). Inducible vectors which may be mentionedare pTet-tTak, pTet-Splice, pcDNA4/TO, pcDNA4/TO /LacZ, pcDNA6/TR,pcDNA4/TO/Myc-His/LacZ, pcDNA4/TO/Myc-His A, pcDNA4/TO/Myc-His B,pcDNA4/TO/Myc-His C, pVgRXR (Invitrogen, Inc.) or the pMAM series(Clontech, Inc.; GenBank Accession No: U02443). These themselves providethe inducible regulatory control element for example for a chemicallyinducible expression.

Vectors for expression in yeast comprise for example pYES2, pYD1,pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, PHIL-D2,PHIL-S1, pPIC3SK, pPIC9K, and PA0815 (Invitrogen, Inc.).

Cloning vectors and techniques for genetic manipulation of ciliates andalgae are known to the skilled worker (WO 98/01572; Falciatore et al.(1999) Marine Biotechnology 1(3):239-251; Dunahay et al. (1995) J Phycol31:10004-1012).

The methods to be used in principle for the transformation of animalcells or of yeast cells are similar to those for “direct” transformationof plant cells. Methods such as calcium phosphate or liposome-mediatedtransformation or else electroporation are preferred in particular.

Various methods and vectors for inserting genes into the genome ofplants and for regenerating plants from plant tissues or plant cells areknown (Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton,Fla.), Chapter 6/7, pp. 71-119 (1993); White F F (1993) Vectors for GeneTransfer in Higher Plants; in: Transgenic Plants, Vol. 1, Engineeringand Utilization, editors: Kung and Wu R, Academic Press, 15-38; Jenes Bet al. (1993) Techniques for Gene Transfer, in: Transgenic Plants, Vol.1, Engineering and Utilization, editors: Kung and R. Wu, Academic Press,pp. 128-143; Potrykus (1991) Annu Rev Plant Physiol Plant Molec Biol42:205-225; Halford N G, Shewry P R (2000) Br Med Bull 56(1):62-73).Those mentioned above are included, for example. In the case of plants,the described methods for the transformation and regeneration of plantsfrom plant tissues or plant cells are used for transient or stabletransformation. Suitable methods are, in particular, protoplasttransformation by polyethylene glycol-induced DNA uptake, calciumphosphate-mediated transformation, DEAE-dextran-mediated transformation,liposome-mediated transformation (Freeman et al. (1984) Plant CellPhysiol. 29:1353ff; U.S. Pat. No. 4,536,475), biolistic methods with thegene gun (“particle bombardment” method; U.S. Pat. No. 5,100,792; EP-A 0444 882; EP-A 0 434 616; Fromm M E et al. (1990) Bio/Technology8(9):833-9; Gordon-Kamm et al. (1990) Plant Cell 2:603),electroporation, incubation of dry embryos in DNA-containing solution,electroporation (EP-A 290 395, WO 87/06614), microinjection (WO92/09696, WO 94/00583, EP-A 0 331 083, EP-A 0 175 966) or other methodsof direct DNA introduction (DE 4 005 152, WO 90/12096, U.S. Pat. No.4,684,611). Physical methods of DNA introduction into plant cells aresurveyed in Oard (1991) Biotech Adv 9:1-11.

In the case of these “direct” transformation methods, no particularrequirements need be met by the plasmid used. Simple plasmids such asthose of the pUC series, pBR322, M13 mp series, pACYC184 etc. can beused. If complete plants are to be regenerated from the transformedcells, it is necessary for an additional selectable marker gene to bepresent on the plasmid.

Besides these “direct” transformation techniques, it is also possible tocarry out a transformation by bacterial infection using agrobacterium(e.g. EP 0 116 718), viral infection using viral vectors (EP 0 067 553;U.S. Pat. No. 4,407,956; WO 95/34668; WO 93/03161) or using pollen (EP 0270 356; WO 85/01856; U.S. Pat. No. 4,684,611).

The transformation is preferably effected using agrobacteria whichcomprise disarmed Ti plasmid vectors, utilizing their natural ability totransfer genes to plants (EP-A 0 270 355; EP-A 0 116 718).

Agrobacterium transformation is widely used for the transformation ofdicotyledons, but is also increasingly being applied to monocotyledons(Toriyama et al. (1988) Bio/Technology 6: 1072-1074; Zhang et al. (1988)Plant Cell Rep 7:379-384; Zhang et al. (1988) Theor Appl Genet76:835-840; Shimamoto et al. (1989) Nature 338:274-276; Datta et al.(1990) Bio/Technology 8: 736-740; Christou et al. (1991) Bio/Technology9:957-962; Peng et al. (1991) International Rice Research Institute,Manila, Philippines 563-574; Cao et al. (1992) Plant Cell Rep11:585-591; Li et al. (1993) Plant Cell Rep 12:250-255; Rathore et al.(1993) Plant Mol Biol 21:871-884; Fromm et al. (1990) Bio/Technology8:833-839; Gordon-Kamm et al. (1990) Plant Cell 2:603-618; D'Halluin etal. (1992) Plant Cell 4:1495-1505; Walters et al. (1992) Plant Mol Biol18:189-200; Koziel et al. (1993) Biotechnology 11:194-200; Vasil I K(1994) Plant Mol Biol 25:925-937; Weeks et al. (1993) Plant Physiol102:1077-1084; Somers et al. (1992) Bio/Technology 10:1589-1594; WO92/14828; Hiei et al. (1994) Plant J 6:271-282).

The strains mostly used for agrobacterium transformation, Agrobacteriumtumefaciens or Agrobacterium rhizogenes comprise a plasmid (Ti or Riplasmid) which is transferred to the plant after agrobacteriuminfection. Part of this plasmid, called T-DNA (transferred DNA), isintegrated into the genome of the plant cell. Alternatively, binaryvectors (mini-Ti plasmids) can also be transferred into plants andintegrated in the genome thereof by agrobacterium.

The use of Agrobacterium tumefaciens for the transformation of plantsusing tissue culture explants is described (inter alia Horsch R B et al.(1985) Science 225:1229ff.; Fraley et al. (1983) Proc Natl Acad Sci USA80: 4803-4807; Bevans et al. (1983) Nature 304:184-187). ManyAgrobacterium tumefaciens strains are able to transfer geneticmaterial—for example the expression cassettes of the invention—such as,for example, the strains EHA101[pEHA101], EHA105[pEHA105],LBA4404[pAL4404], C58C1[pMP90] and C58C1[pGV2260] (Hood et al. (1993)Transgenic Res 2:208-218; Hoekema et al. (1983) Nature 303:179-181;Koncz and Schell (1986) Gen Genet 204:383-396; Deblaere et al. (1985)Nucl Acids Res 13: 4777-4788).

On use of agrobacteria, the expression cassette must be integrated intospecific plasmids either into a shuttle, or intermediate, vector or intoa binary vector. Binary vectors, which are able to replicate both in E.coli and in agrobacterium, are preferably used. They normally comprise aselection marker gene and a linker or polylinker, flanked by the rightand left T-DNA border sequence. They can be transformed directly intoagrobacterium (Holsters et al. (1978) Mol Gen Genet 163:181-187). Theagrobacterium acting as host organism in this case should alreadycomprise a plasmid having the vir region. This is necessary for transferof the T-DNA into the plant cell. An agrobacterium transformed in thisway can be used to transform plant cells. The use of T-DNA fortransforming plant cells has been intensively investigated and described(EP-A 0 120 516; Hoekema, In: The Binary Plant Vector System,Offsetdrukkerij Kanters B. V., Alblasserdam, Chapter V; An et al. (1985)EMBO J 4:277-287). Various binary vectors are known, and some of themare commercially available, such as, for example, pBI101.2 or pBIN19(Clontech Laboratories, Inc. USA; Bevan et al. (1984) Nucl Acids Res12:8711), pBinAR, pPZP200 or pPTV.

Agrobacteria transformed with such a vector can then be used in a knownmanner for transforming plants, especially crop plants such as, forexample, oilseed rape, by for example bathing wounded leaves or piecesof leaf in a solution of agrobacteria and then cultivating in suitablemedia. Transformation of plants by agrobacteria is described (White F F(1993) Vectors for Gene Transfer in Higher Plants; in Transgenic Plants,Vol. 1, Engineering and Utilization, edited by SD Kung and R Wu,Academic Press, pp. 15-38; Jenes B et al. (1993) Techniques for GeneTransfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization,edited by S. D. Kung and R. Wu, Academic Press, pp. 128-143; Potrykus(1991) Annu Rev Plant Physiol Plant Molec Biol 42:205-225). Transgenicplants which have integrated the expression systems of the inventiondescribed above can be regenerated in a known manner from thetransformed cells of the wounded leaves or pieces of leaf.

Stably transformed cells (i.e. those which have integrated theintroduced DNA into the DNA of the host cell) can be selected fromuntransformed ones if a selectable marker is a constituent of theintroduced DNA. Any gene able to confer a resistance (see above) to abiocide (e.g. an antibiotic or herbicide, see above) can act as marker,for example. Transformed cells which express such a marker gene are ableto survive in the presence of concentrations of a corresponding biocidewhich kill an untransformed wild type. The selection marker permits theselection of transformed cells from untransformed ones (McCormick et al.(1986) Plant Cell Reports 5:81-84). The resulting plants can be grownand crossed in the usual way. Two or more generations should becultivated in order to ensure that the genomic integration is stable andheritable.

As soon as a transformed plant cell has been produced, it is possible toobtain a complete plant by using methods known to the skilled worker.These entail, for example, starting from callus cultures, single cells(e.g. protoplasts) or leaf disks (Vasil et al. (1984) Cell Culture andSomatic Cell Genetics of Plants, Vol I, II and III, LaboratoryProcedures and Their Applications, Academic Press; Weissbach andWeissbach (1989) Methods for Plant Molecular Biology, Academic Press).The formation of shoot and root from these still undifferentiated calluscell masses can be induced in a known manner. The resulting shoots canbe planted out and grown. Corresponding methods are described (Fennellet al. (1992) Plant Cell Rep. 11: 567-570; Stoeger et al (1995) PlantCell Rep. 14:273-278; Jahne et al. (1994) Theor Appl Genet 89:525-533).

The effectiveness of expression of the transgenically expressed nucleicacids can be estimated for example in vitro by shoot-meristempropagation using one of the selection methods described above. Inaddition, a change in the type and level of expression of a target gene,and the effect on the phenotype of the plant can be tested on testplants in glasshouse tests.

A further aspect of the invention relates to transgenic organismstransformed with at least one expression cassette of the invention orone vector of the invention, and cells, cell cultures, tissues,parts—such as, for example, in the case of plant organisms leaves, rootsetc.—or propagation material derived from such organisms.

By organism, starting or host organisms are meant prokaryotic oreukaryotic organisms such as, for example, microorganisms or plantorganisms. Preferred microorganisms are bacteria, yeasts, algae orfungi.

Preferred bacteria are bacteria of the genus Escherichia, Erwinia,Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas, Bacillus orcyanobacteria, for example of the genus Synechocystis and furtherbacterial genera described in Brock Biology of Microorganisms EighthEdition on pages A-8, A-9, A10 and A11.

Microorganisms which are particularly preferred are those able to infectplants and thus transfer the constructs of the invention. Preferredmicroorganisms are those of the genus Agrobacterium and especially ofthe species Agrobacterium tumefaciens. Particularly preferredmicroorganisms are those able to produce toxins (e.g. botulinus toxin),pigments (e.g. carotenoids or flavonoids), antibiotics (e.g.penicillin), phenylpropanoids (e.g. tocopherol), polyunsaturated fattyacids (e.g. arachidonic acid) or vitamins (e.g. vitamin B12).

Preferred yeasts are Candida, Saccharomyces, Hansenula or Pichia.

Preferred fungi are Aspergillus, Trichoderma, Ashbya, Neurospora,Fusarium, Beauveria or further fungi described in Indian Chem Engr.Section B. Vol 37, No. 1,2 (1995) on page 15, table 6.

Host or starting organisms preferred as transgenic organisms are inparticular plant organisms.

“Plant organism or cells derived therefrom” means in general every cell,tissue, part or propagation material (such as seeds or fruits) of anorganism capable of photosynthesis. Included for the purposes of theinvention are all genera and species of higher and lower plants of theplant kingdom. Annual, perennial, monocotyledonous and dicotyledonousplants are preferred.

“Plant” means for the purposes of the invention all genera and speciesof higher and lower plants of the plant kingdom. The term includes themature plants, seeds, shoots and seedlings, and parts derived therefrom,propagation material (for example tubers, seeds or fruits), plantorgans, tissues, protoplasts, callus and other cultures, for examplecell or callus cultures, and all other types of groupings of plant cellsto functional or structural units. Mature plants means plants at anystage of development beyond seedling. Seedling means a young, immatureplant at an early stage of development.

Plant organisms for the purposes of the invention are additionallyfurther photosynthetically active organisms such as, for example, algae,cyanobacteria and mosses. Preferred algae are green algae, such as, forexample, algae of the genus Haematococcus, Phaedactylum tricornatum,Volvox or Dunaliella. Synechocystis, Chlamydomonas and Scenedesmus areparticularly preferred.

Particularly preferred for the purposes of the method of the inventionare plant organisms selected from the group of flowering plants (PhylumAnthophyta “angiosperms”). All annual and perennial, monocotyledonousand dicotyledonous plants are comprised. The plant is preferablyselected from the following plant families: Amaranthaceae, Asteraceae,Brassicaceae, Caryophyllaceae, Chenopodiaceae, Compositae, Cruciferae,Cucurbitaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae,Linaceae, Malvaceae, Rosaceae, Rubiaceae, Saxifragaceae,Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceaeand Umbelliferae.

The invention is very particularly preferably applied to dicotyledonousplant organisms. Preferred dicotyledonous plants are in particularselected from the dicotyledonous crop plants such as, for example thefollowing

-   1) Category: Dicotyledonae (dicotyledons). Preferred families:    -   Aceraceae (maples)    -   Cactaceae (cacti)    -   Rosaceae (roses, apples, almonds, strawberries)    -   Salicaceae (willows)    -   Asteraceae (compositae) especially the genus Lactuca, very        especially the species sativa (lettuce), and sunflower,        dandelion, Tagetes or Calendula and many others,    -   Cruciferae (Brassicaceae), especially the genus Brassica, very        especially the species napus (oilseed rape), campestris (beet),        oleracea (e.g. cabbage, cauliflower or broccoli and other        brassica species); and of the genus Arabidopsis, very especially        the species thaliana, and cress, radish, canola and many others,    -   Cucurbitaceae such as melon, pumpkin squash, cucumber or        zucchini and many others,    -   Leguminosae (Fabaceae) especially the genus Glycine, very        especially the species max (soybean), soya and alfalfa, pea,        beans, lupin or peanut and many others,

Malvaceae, especially mallow, cotton, edible marshmallow, hibiscus andmany others,

Rubiaceae, preferably of the subclass Lamiidae such as, for example,Coffea arabica or Coffea liberica (coffee bush) and many others,

Solanaceae, especially the genus Lycopersicon, very especially thespecies esculentum (tomato) and the genus Solanum, very especially thespecies tuberosum (potato) and melongena (eggplant) and the genusCapsicum, very especially the species. annuum (paprika), and tobacco,petunia and many others,

Sterculiaceae, preferably of the subclass Dilleniidae such as, forexample, Theobroma cacao (cocoa bush) and many others,

Theaceae, preferably of the subclass Dilleniidae such as, for example,Camellia sinensis or Thea sinensis (tea bush) and many others,

Umbelliferae (Apiaceae), especially the genus Daucus (very especiallythe species carota (carrot), Apium (very especially the speciesgraveolens dulce (celeriac)), and parsley and many others; and linum,hemp, flax, spinach, carrot, sugarbeet and the various tree, nut andvine species, especially banana and kiwi fruit.

However, in addition, monocotyledonous plants are also suitable. Theseare preferably selected from the monocotyledonous crop plants such as,for example the families

-   -   Arecaceae (palms)    -   Bromeliaceae (pineapple, spanish moss)    -   Cyperaceae (sedges)    -   Liliaceae (lilies, tulips, hyacinths, onions, garlic)    -   Orchidaceae (orchids)    -   Poaceae (grasses, bamboos, corn, sugarcane, wheat)    -   Iridaceae (buckwheat, gladioli, crocuses)

Very particular preference is given to Gramineae such as rice, corn,wheat or other cereal species such as barley, sorghum and millet, rye,triticale or oats, and the sugarcane, and all species of grasses.

Within the framework of the expression cassette of the invention,expression of a particular nucleic acid may, through a promoter havingspecificity for the nonreproductive organs of the flower, lead to theformation of sense RNA, antisense RNA or double-stranded RNA in the formof an inverted repeat (dsRNAi). The sense RNA can subsequently betranslated into particular polypeptides. It is possible with theantisense RNA and dsRNAi to down regulate the expression of particulargenes.

The method of gene regulation by means of double-stranded RNA(“double-stranded RNA interference”; dsRNAi) has been described inanimal and plant organisms many times (e.g. Matzke M A et al. (2000)Plant Mol Biol 43:401-415; Fire A et al (1998) Nature 391:806-811; Wo99/32619; WO 99/53050; WO 00/68374; WO 00/44914; WO 00/44895; WO00/49035; WO 00/63364). Express reference is made to the processes andmethods described in the citations indicated.

The specificity of the expression constructs and vectors of theinvention for flowers of plants is particularly advantageous. The flowerhas a function in attracting beneficial insects through incorporation ofpigments or synthesis of volatile chemicals.

The natural defense mechanisms of the plant, for example againstpathogens, are often inadequate. Introduction of foreign genes fromplants, animals or microbial sources may enhance the defenses. Examplesare protection against insect damage to tobacco through expression ofthe Bacillus thuringiensis endotoxin (Vaeck et al. (1987) Nature328:33-37) or protection of tobacco from fungal attack throughexpression of a chitinase from beans (Broglie et al. (1991) Science254:1194-1197).

Cold spells during the flowering period lead to considerable crop lossesevery year. Targeted expression of protective proteins specifically inthe flowering period may provide protection.

For such genetic engineering approaches to be highly efficient it isadvantageous for there to be concentrated expression of the appropriatenucleic acid sequence to be expressed transgenically in particular inthe petals of the flower. Constitutive expression in the whole plant maymake the effect problematic, for example through dilution, or impair thegrowth of the plant or the quality of the plant product. In addition,there may through constitutive expression be increased switching-off ofthe transgene (“gene silencing”).

Promoters having specificity for the flower are advantageous in thisconnection. The skilled worker is aware of a large number of proteinswhose recombinant expression in the flower is advantageous. The skilledworker is also aware of a large number of genes through whichadvantageous effects can likewise be achieved through repression orswitching-off thereof by means of expression of a correspondingantisense RNA. Non-restrictive examples of advantageous effects whichmay be mentioned are: achieving resistance to abiotic stress factors(heat, cold, drought, increased moisture, environmental toxins, UVradiation) and biotic stress factors (pathogens, viruses, insects anddiseases), improving the properties of human and animal foods, improvingthe growth rate or the yield, achieving a longer or earlier floweringperiod, altering or enhancing the scent or the coloring of the flowers.Non-restrictive examples of the nucleic acid sequences or polypeptideswhich can be employed in these applications and which may be mentionedare:

-   1. Improved UV protection of the flowers of plants through    alteration of the pigmentation through expression of particular    polypeptides such as enzymes or regulators of flavonoid biosynthesis    (e.g. chalcone synthases, phenylalanine ammonia-lyases), of DNA    repair (e.g. photolyases; Sakamoto A et al. (1998) DNA Seq    9(5-6):335-40), of isoprenoid biosynthesis (e.g.    deoxyxylulose-5-phosphate synthases), of IPP synthesis or of    carotenoid biosynthesis (e.g. phytoene synthases, phytoene    desaturases, lycopene cyclases, hydroxylases or ketolases).    Preference is given to nucleic acids which code for the Arabidopsis    thaliana chalcone synthase (GenBank Acc. No.: M20308), the    Arabidopsis thaliana 6-4 photolyase (GenBank Acc. No.:BAB00748) or    the Arabidopsis thaliana blue light photoreceptor/photolyase homolog    (PHHl) (GenBank Acc. No.: U62549) or functional equivalents thereof.-   2. Improved protection of the flower of plants from abiotic stress    factors such as drought, heat or cold, for example through    overexpression of the antifreeze polypeptides (e.g. from    Myoxocephalus scorpius; WO 00/00512), of the Arabidopsis thaliana    transcription activator CBF1, glutamate dehydrogenases (WO 97/12983,    WO 98/11240), of a late embryogenesis gene (LEA), for example from    barley (WO 97/13843), calcium-dependent protein kinase genes (WO    98/26045), calcineurins (WO 99/05902), farnesyl transferases (WO    99/06580; Pei Z M et al. (1998) Science 282:287-290), ferritin (Deak    M et al. (1999) Nature Biotechnology 17:192-196), oxalate oxidase    (WO 99/04013; Dunwell J M (1998) Biotechnology and Genetic    Engeneering Reviews 15:1-32), DREBlA factor (dehydration response    element B 1A; Kasuga M et al. (1999) Nature Biotechnology    17:276-286), genes of mannitol or trehalose synthesis (e.g.    trehalose-phosphate synthases; trehalose-phosphate phosphatases, WO    97/42326); or through inhibition of genes such as of trehalase (WO    97/50561). Particular preference is given to nucleic acids which    code for the Arabidopsis thaliana transcriptional activator CBF1    (Gen-Bank Acc. No.: U77378) or the antifreeze protein from    Myoxocephalus octodecemspinosus (GenBank Acc. No.: AF306348) or    functional equivalents thereof.

Achieving resistance for example to fungi, insects, nematodes anddiseases through targeted secretion or accumulation of certainmetabolites or proteins in the flower. Examples which may be mentionedare glucosinolates (nematode defense), chitinases or glucanases andother enzymes which destroy the cell wall of parasites,ribosome-inactivating proteins (RIPs) and other proteins of the plantresistance and stress response, like those induced on injury ormicrobial attack of plants or chemically by, for example, salicylicacid, jasmonic acid or ethylene, lysozymes from non-plant sources suchas, for example, T4 lysozyme or lysozme from various mammals,insecticidal proteins such as Bacillus thuringiensis endotoxin,α-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor),glucanases, lectins (e.g. phytohemagglutinin, snowdrop lectin, wheatgermagglutinin), RNAses or ribozymes. Particular preference is given tonucleic acids which code for the chit42 endochitinase from Trichodermaharzianum (GenBank Acc. No.: S78423) or for the N-hydroxylating,multifunctional cytochrome P-450 (CYP79) from Sorghum bicolor (GenBankAcc. No.: U32624) or functional equivalents thereof.

-   4. Achieving defense against or attraction of insects, for example    through increased release of volatile scents or messengers through,    for example, enzymes of terpene biosynthesis.-   5. Achieving an ability to store in flower tissues which normally    comprise no storage proteins or lipids, with the aim of increasing    the yield of these substances, e.g. by expression of an acetyl-CoA    carboxylase or of enzymes for esterification of metabolites.    Preference is given to nucleic acids which code for the Medicago    sativa acetyl-CoA carboxylase (Accase) (GenBank Acc. No.: L25042) or    functional equivalents thereof.-   6. Expression of transport proteins which improve the uptake of    metabolites, nutrients or water into the flower and thus optimize    flower growth, metabolite composition or yield, for example through    expression of an amino acid transporter which increases the rate of    uptake of amino acids, or of a monosaccharide transporter which    promotes the uptake of sugars. Preference is given to nucleic acids    which code for the Arabidopsis thaliana cationic amino acid    transporter (GenBank Acc. No.: X92657) or for the Arabidopsis    thaliana monosaccharide transporter (Gen-Bank Acc. No.: AJ002399) or    functional equivalents thereof.-   7. Expression of genes which bring about an accumulation of fine    chemicals, such as of tocopherols, tocotrienols, phenylpropanoids,    isoprenoids or carotenoids, in the flower. Examples which may be    mentioned are the deoxyxylulose-5-phosphate synthases, phytoene    synthases, lycopene β-cyclases and the β-carotene ketolases.    Preference is given to nucleic acids which code for the Haematoccus    pluvialis NIES-144 (Acc. No. D45881) ketolase or functional    equivalents thereof.-   8. Modification of wax ester formation or of the composition of the    deposited oligosaccharides to improve protection against    environmental effects or to improve digestibility on use in animal    or human foods. An example which may be mentioned is overexpression    of endo-xyloglucan transferase. Preference is given to nucleic acids    which code for the Arabidopsis thaliana endo-xyloglucan transferase    (EXGT-Al) (Gen-Bank Acc. No.:AF163819) or functional equivalents    thereof.-   9. Expression of genes, DNA binding proteins, dsRNA and antisense    constructions for altering the flower morphology, the time of    flowering and the flower senescence, and the flower metabolism.    Preference is given to constructions which increase the number of    petals, e.g. through down regulation of AGAMOUS and its homologous    genes (Yanofsky M F et al. (1990) Nature 346:35-39), make the time    of flowering earlier, e.g. through down regulation of FLOWERING    LOCUS C (FLC) (Tadege M et al. (2001) Plant J 28(5):545-53) or    later, e.g. through overexpression of FLC, and delay senescence,    e.g. through conferring a flower-specific ethylene insensitivity.-   10. Generation of sterile plants by preventing pollenation and/or    germination by means of the expression of a suitable inhibitor, for    example of a toxin, in flowers.-   11. Production of nutraceuticals such as, for example    -   a) carotenoids and/or phenylpropanoids e.g. through optimization        of the flowers' own metabolic pathways, e.g. through expression        of enzymes and regulators of isoprenoid biosynthesis. Preference        is given to nucleic acids which code for the Arabidopsis        thaliana chalcone synthase (GenBank Acc. No.: M20308), the        Arabidopsis thaliana 6-4 photolyase (GenBank Acc. No.:BAB00748)        or the Arabidopsis thaliana blue light photoreceptor/photolyase        homolog (PHHl) (GenBank Acc. No.: U62549) or functional        equivalents thereof. Preference is likewise given to nucleic        acids which code for enzymes and regulators of isoprenoid        biosynthesis such as the deoxyxylulose-5-phosphate synthases and        of carotenoid biosynthesis such as the phytoene synthases,        lycopene cyclases and ketolases, such as of tocopherols,        tocotrienols, phenylpropanoids, isoprenoids or carotenoids, in        the flower. Examples which may be mentioned are the        deoxyxylulose-5-phosphate synthases, phytoene synthases,        lycopene cyclases and the carotene ketolases. Particular        preference is given to nucleic acids which code for the        Haematoccus pluvialis, NIES-144 (Acc. No. D45881) ketolase or        functional equivalents.    -   b) polyunsaturated fatty acids such as, for example, arachidonic        acid or EPA (eicosapentaenoic acid) or DHA (docosahexaenoic        acid) through expression of fatty acid elongases and/or        desaturases or production of proteins having improved        nutritional value, such as, for example, having a high content        of essential amino acids (e.g. the methionine-rich 2S albumin        gene of the Brazil nut). Preference is given to nucleic acids        which code for the Bertholletia excelsa methionine-rich 2S        albumin (GenBank Acc. No.: AB044391), the Physcomitrella patens        A6-acyl lipid desaturase (GenBank Acc. No.: AJ222980; Girke et        al. (1998) Plant J 15:39-48), the Mortierella alpina        Δ6-desaturase (Sakura-dani et al 1999 Gene 238:445-453), the        Caenorhabditis elegans Δ5-desaturase (Michaelson et al. (1998)        FEBS Letters 439:215-218), the Caenorhabditis elegans        Δ5-fatty-acid desaturase (des-5) (GenBank Acc. No.: AF078796),        the Mortierella alpina Δ5-desaturase (Michaelson et al. J Biol        Chem 273:19055-19059), the Caenorhabditis elegans Δ6-elongase        (Beaudoin et al. (2000) Proc Natl. Acad. Sci. 97:6421-6426), the        Physcomitrella patens Δ6-elongase (Zank et al. (2000,)        Biochemical Society Transactions 28:654-657) or functional        equivalents thereof.-   12. Production of pharmaceuticals such as, for example, antibodies,    vaccines, hormones and/or antibiotics as described, for example, in    Hood E E & Jilka J M (1999) Curr Opin Biotechnol 10(4):382-6; Ma J K    & Vine N D (1999) CurrTop Microbiol Immunol 236:275-92.

Further examples of advantageous genes are mentioned for example inDunwell J M (2000) Transgenic approaches to crop improvement. J Exp Bot.51 Spec No:487-96.

A further aspect of the invention relates to the use of the transgenicorganisms of the invention described above, and of the cells, cellcultures, parts—such as, for example, in the case of transgenic plantorganisms roots, leaves etc.—and transgenic propagation material such asseeds or fruits, derived therefrom for producing human or animal foods,pharmaceuticals or fine chemicals.

Preference is further given to a method for the recombinant productionof pharmaceuticals or fine chemicals in host organisms, where a hostorganism is transformed with one of the expression cassettes describedabove, and this expression cassette comprises one or more structuralgenes which code for the desired fine chemical, or catalyze thebiosynthesis thereof, the transformed host organism is cultivated, andthe desired fine chemical is isolated from the cultivation medium. Thismethod can be applied widely to fine chemicals such as enzymes,vitamins, amino acids, sugars, fatty acids, natural and syntheticflavorings, aromatizing substances and colorants. Production oftocopherols and tocotrienols, and carotenoids such as, for example,astaxanthin is particularly preferred. Cultivation of the transformedhost organisms and isolation from the host organisms or from thecultivation medium take place by methods known to the skilled worker.The production of pharmaceuticals such as, for example, antibodies orvaccines is described in Hood E E & Jilka J M (1999) Curr OpinBiotechnol 10 (4)382-6; Ma J K & Vine N D (1999) Curr Top MicrobiolImmunol 236:275-92. Sequences  1. SEQ ID NO: 1 2051 bp fragment ofpromoter (and if appropriate 5′ untrans- lated region of the Arabidopsisthaliana gene locus At3g01980 (76L promoter)  2. SEQ ID NO: 2 2192 bpfragment of promoter (and if appropriate 5′ untrans- lated region of theArabidopsis thaliana gene locus At1g63140 (84L promotor)  3. SEQ ID NO:3 Functionally equivalent frag- ment (1045 bp) of promoter (and ifappropriate 5′ untranslated region of Arabidopsis thaliana gene locusAt3g01980 (76S promoter)  4. SEQ ID NO: 4 Functionally equivalent frag-ment (1109 bp) of promoter (and if appropriate 5′ untranslated region ofArabidopsis thaliana gene locus At1g63140 (84L promoter)  5. Seq ID No:5 Oligonucleotide primer 76sSmaI 5′-CCCGGGTGCCAAAGTAACTCTTTAT-3′  6. SeqID No: 6 Oligonucleotide primer 76assSalI5′-GTCGACAGGTGCATGACCAAGTAAC-3′  7. Seq ID No: 7 Oligonucleotide primer76aslSalI 5′-GTCGACTATCCTCTGCGCAATGAAT-3′  8. Seq ID No: 8Oligonucleotide primer 84sSmaI 5′-CCCGGGAAATCGAGAAAGATAGGTA-3′  9. SeqID No: 9 Oligonucleotide primer 84assSalI5′-GTCGACAAAGGGTTATAGGAGACTG-3′ 10. Seq ID No: 10 Oligonucleotide primer84aslSalI 5′-GTCGACCATGTTTCAGAGGATATGT-3′ 11. SEQ ID NO: 11 Nucleic acidsequence (cDNA) encoding the gene product of the Arabidopsis thalianagene locus At3g01980 12. SEQ ID NO: 12 Amino acid sequence encoding thegene product of the Arabidopsis thaliana gene locus At3g01980 13. SEQ IDNO: 13 Nucleic acid sequence (cDNA) encoding the gene product of theArabidopsis thaliana gene locus At1g63140 14. SEQ ID NO: 14 Amino acidsequence encoding the gene product of the Arabidopsis thaliana genelocus At1g63140 15. SEQ ID NO: 15 Nucleic acid sequence (cDNA) encodingthe oilseed rape homolog (H2) of the At3g01980 gene product 16. SEQ IDNO: 16 Amino acid sequence encoding the oilseed rape homolog (H2) of theAt3g01980 gene product 17. SEQ ID NO: 17 Nucleic acid sequence (cDNA)encoding the oilseed rape homolog (H3) of the At3g01980 gene product 18.SEQ ID NO: 18 Amino acid sequence encoding the oilseed rape homolog (H3)of the At3g01980 gene product 19. SEQ ID NO: 19 Nucleic acid sequence(cDNA) encoding the oilseed rape homolog (H4) of the At1g63140 geneproduct 20. SEQ ID NO: 20 Amino acid sequence encoding the oilseed rapehomolog (H4) of the At1g63140 gene product 21. SEQ ID NO: 21 Nucleicacid sequence (cDNA) encoding the oilseed rape homolog (H5) of theAt1g63140 gene product 22. SEQ ID NO: 22 Amino acid sequence encodingthe oilseed rape homolog (H5) of the At1g63140 gene product 23.-32 SEQID NO: 23 bis 32: Sequence motifs for proteins with specific expres-sion in the nonreproductive floral tissues. 33. Seq ID No: 33Oligonucleotide primer GUS for 5′-cac ttt tcc cgg caa taa cat- 3′ 34.Seq ID No: 34 Oligonucleotide primer GUS rev 5′-atc agg aag tga tgg agcatc- 3′ 35. Seq ID No: 35 Oligonucleotide primer TUB for 5′-gac cct gtccca cct cca a-3′ 36. Seq ID No: 36 Oligonucleotide primer TUB rev 5′-tgagaa ctg cga ttg ttt gca- 3′Figures

The general abbreviations used in the following figures have thefollowing meaning:

-   -   GUS: reporter gene (bacterial β-glucuronidase)    -   Int: Intron    -   NosT: nopaline synthase (NOS) terminator sequence    -   NptII: BASTA resistance    -   NosP: nopaline synthase (NOS) promoter sequence    -   AadA: bacterial spectinomycin resistance

-   1. FIG. 1: Diagrammatic representation of the vector pSUN3-76L-GUS.    Further abbreviations have the following meaning:    -   76L: 76L promoter of SEQ ID NO:1

-   2. FIG. 2: Diagrammatic representation of the vector pSUN3-76S-GUS.    Further abbreviations have the following meaning:    -   76S: 76S promoter of SEQ ID NO: 3

-   3. FIG. 3: Diagrammatic representation of the vector pSUN3-84L-GUS.    Further abbreviations have the following meaning:    -   84L: 84L promoter of SEQ ID NO: 2

-   4. FIG. 4: Diagrammatic representation of the vector pSUN3-84S-GUS.    Further abbreviations have the following meaning:    -   84S: 84S promoter of SEQ ID NO: 4

-   5. FIG. 5: Diagrammatic representation of the vector pSUN5-P76-GUS.    Further abbreviations have the following meaning:    -   P76: 76S promoter of SEQ ID NO: 3

-   6. FIG. 6: The expression patterns of the promoters 76 (A) and    84 (B) in the flower of Arabidopsis thaliana are shown. White/pale    gray areas indicate tissues without promoter activity.

-   7. FIG. 7: The expression patterns of the promoters 76 (A) and    84 (B) in the inflorescences and leaves of Arabidopsis thaliana are    shown. White/pale gray areas indicate tissues without promoter    activity.

-   8. FIG. 8: The resolution of the promoter activity of the promoter    76 over time during the floral development of Arabidopsis thaliana    is shown. A: β-glucuronidase mRNA quantities in six stages of the    floral development (P1 to P6) of Arabidopsis thaliana for the    promoter 76. The data were determined by means of quantiative “real    time” PCR and standardized with the 1 kb P76 promoter during    flowering stage P4 (to this end, the value in question was set equal    to 1). B: The points in time of the development of the Arabidopsis    flowers which correspond to the flowering stages P1 to P6 are shown.

-   9. FIG. 9: The resolution of the promoter activity of the promoter    84 over time during the floral development of Arabidopsis thaliana    is shown. A: β-glucuronidase mRNA quantities in six stages of the    floral development (P1 to P6) of Arabidopsis thaliana for the    promoter 84. the data were determined by means of quantiative “real    time” PCR and standardized with the during flowering stage P2 (to    this end, the value in question was set equal to 1). B: The points    in time of the development of the Arabidopsis flowers which    correspond to the flowering stages P1 to P6 are shown.

-   10. FIG. 10: A resolution of the promoter activity of the promoter    76 over time during the floral development of Tagetes erecta are    shown. A: β-glucuronidase enzyme activity (shown in pmol of methyl    umbelliferon/min/mg protein) during six stages of the floral    development (P1 to P6) of Tagetes erecta for the promoter 76. In    each case 3 individual measurements are shown (black bars, gray    bars, white bars).    -   B: The points in time of the development of the Tagetes flowers        which correspond to the flowering stages P1 to P6 are shown.

-   11. FIG. 11: The resolution of the promoter activity of the promoter    76 over time during the floral development of Tagetes erecta is    shown. A: β-glucuronidase mRNA quantities in six stages of the    floral development (P1 to P6) of Tagetes erecta for the promoter 76.    The data were determined by means of quantiative “real time” PCR and    standardized with the during flowering stage P4 (to this end, the    value in question was set equal to 1). B: The points in time of the    development of the Tagetes flowers which correspond to the flowering    stages P1 to P6 are shown.

-   12. FIG. 12: Protein sequence alignment between the SEQ ID NO: 12    amino acid sequence (cDNA) encoding the gene product of the    Arabidopsis thaliana gene locus At3g01980 and a cDNA clone from a    Brassica napus floral cDNA library.

-   13. FIG. 13: Protein sequence alignment between the SEQ ID NO: 14    amino acid sequence (cDNA) encoding the gene product of the 20    Arabidopsis thaliana gene locus At1g63140 and a cDNA clone from a    Brassica napus floral cDNA library.

-   14. FIG. 14: diagrammatic representation of the inverse PCR    (“iPCR”). For the “iPCR”, genomic DNA of a target organism having    the promoter sequence to be isolated is completely digested with a    given restriction enzyme, and then the individual fragments are    religated, i.e. connected together to form a circular molecule, in a    diluted mixture. The large number of resulting circular DNA    molecules includes those comprising the known sequence (i.e. the    sequence coding for a homologous protein). The circular molecule can    be amplified, starting therefrom, by means of PCR using a primer    pair in which both primers are able to anneal to the known sequence    segment. Abbreviations: P—promoter sequence; CR—coding region;    L—ligation site; PCR—polymerase chain reaction. Arrows represent the    binding site of potential oligonucleotide primers in the area of the    coding region.

EXAMPLES

General Methods:

Oligonucleotides can be chemically synthesized for example in a knownmanner by the phosphoramidite method (Voet & Voet (1995), 2^(nd)edition, Wiley Press New York, pages 896-897). The cloning steps carriedout for the purposes of the present invention, such as, for example,restriction cleavages, agarose gel electrophoresis, purification of DNAfragments, transfer of nucleic acids to nitrocellulose and nylonmembranes, linkage of DNA fragments, transformation of E. coli cells,culturing of bacteria, replication of phages and sequence analysis ofrecombinant DNA, are carried out as described in Sambrook et al. (1989)Cold Spring Harbor Laboratory Press; ISBN 0-87969-309-6. Recombinant DNAmolecules are sequenced by the method of Sanger (Sanger et al. (1977)Pro Natl Acad Sci USA 74:5463-5467) using an ABI laser fluorescence DNAsequencer.

To generate transgenic Arabidopsis plants, Agrobacterium tumefaciens(strain C58C1 pMP90) is transformed with various promoter GUS vectorconstructs. The agrobacterial strains are subsequently used for thegeneration of transgenic plants. To this end, an individual transformedAgrobacterium colony is incubated in a 4 ml culture (medium: YEB mediumsupplemented with 50 μg/ml kanamycin and 25 μg/ml of rifampicinovernight at 28° C. This culture is subsequently used to inoculate a 400ml culture in the same medium, and this culture is incubated overnight(28° C., 220 rpm) and spun down (GSA rotor, 8000 rpm, 20 min). Thepellet is resuspended in infiltration medium (1/2 MS medium; 0.5 g/lMES, pH 5.8; 50 g/l sucrose). The suspension is introduced into a plantbox (Duchefa) and 100 ml of SILVET L-77 (polyalkylene oxide-modifiedheptamethyltrisiloxane; Osi Special-ties Inc., Cat. P030196) was addedto a final concentration of 0.02%. In a desiccator, the plant boxtogether with 8 to 12 plants is exposed to a vacuum for 10 to 15minutes, followed by spontaneous aeration. This is repeated 2 to 3times. Thereafter, all the plants are planted into plant pots containingmoist soil and grown under long-day conditions (illumination for 16hours) (daytime temperature 22 to 24° C., nighttime temperature 19° C.;relative atmospheric humidity 65%). The seeds are harvested after 6weeks.

Example 1 Growth Conditions of the Plants for Tissue-Specific RT-PCRAnalysis

To obtain 4- or 7-day-old seedlings, in each case approximately 400seeds (Arabidopsis thaliana ecotype Columbia) are surface-sterilized for2 minutes with an 80% strength ethanol solution, treated for 5 minuteswith sodium hypochlorite solution (0.5% v/v), washed three times withdistilled water and incubated at 4° C. for 4 days to ensure uniformgermination. Thereafter, the seeds are incubated on Petri dishescomprising MS medium (Sigma M5519) with addition of 1% sucrose, 0.5 g/lMES (Sigma M8652), 0.8% Difco-Bacto agar (Difco 0140-01), pH 5.7. Theseedlings are grown in a 16-hour-light/8-hour-dark photoperiod (Philips58W/33 white-light lamps) at 22° C. and harvested after 4 and 7 days,respectively, after the beginning of the germination phase.

To obtain roots, 100 seeds are sterilized as described above, incubatedfor 4 days at 4° C. and then grown in 250 ml flasks comprising MS medium(Sigma M5519) with addition of a further 3% sucrose and 0.5 g/l MES(Sigma M8652), pH 5.7. The seedlings are grown in a16-hour-light/8-hour-dark photoperiod (Philips 58W/33 white-light lamps)at 22° C., 120 rpm, and harvested after 3 weeks. For all other plantorgans which are used, the seeds are sown on standard soil (type VM,Manna-Italia, Via S. Giacomo 42, 39050 San Giacomo/Laives, Bolzano,Italy), incubated for 4 days at 4° C. to ensure uniform germination andthen grown in a 16-hour-light/8-hour-dark photoperiod (OSRAM Lumi-luxDaylight 36W/12 fluorescent tubes) at 22° C. Young rosette leaves areharvested in the 8-leaf stage (after 3 weeks), and mature rosette leavesare harvested after 8 weeks shortly before stems are formed.Inflorescences (apices) of the shooting stems are harvested shortlyafter shooting. Stems, stem leaves and flower buds are harvested atdevelopmental stage 12 (Bowmann J (ed.), Arabidopsis, Atlas ofMorphology, Springer New York, 1995) prior to stamen development. Openedflowers are harvested at stage 14 immediately after stamen development.Wilting flowers are harvested at stage 15 to 16. The green and yellowpods which were used were 10 to 13 mm in length.

Example 2 RNA Extraction and cDNA Synthesis

Total RNA is isolated from the plant organs described in Example 1 atvarious points in time of the development, as described (Prescott A,Martin C (1987) Plant Mol Biol Rep 4:219-224). The reverse-transcriptasepolymerase chain reaction (RT-PCR) is used to detect the cDNA of thegene transcripts of At3g01980 and At1G63140. Prior to cDNA synthesis,all RNA samples are treated with DNasel (15 units, Boehringer,Mannheim). The first-strand cDNA synthesis is carried out starting from6 μg of total RNA with an oligo-(dT) primer and RT Superscript™ IIenzyme (300 units) following the manufacturer's instructions in a totalvolume of 20 μl (Life Technologies, Gaithersburg, Md.). To this end, 150ng of “Random Hexamer Primer” are added in a final volume of 12 μl. Themixture is heated for 10 minutes at 70° C. and subsequently immediatelycooled on ice. Then, 4 μl of the 5× first-strand buffer, 2 μl of 0.1 MDTT, 1 μl of 10 mM dNTP-mix (in each case 10 mM dATP, dCTP, dGTP anddTTP) and RNase inhibitor (5 units, Böhringer Mannheim) are added. Themixture is heated for 2 minutes at 42° C., RT Superscript™ II enzyme(300 units, Life Technologies) is added, and the mixture is incubatedfor 50 minutes at 42° C.

Example 3 Detection of the Tissue-Specific Expression

To determine the properties of the promoter and to identify theessential elements thereof, which account for its tissue specificity, itis necessary to place the promoter itself and various fragments thereofbefore what is known as a reporter gene, which makes possible adetermination of the expression activity. An example which may bementioned is the bacterial β-glucuronidase (Jefferson et al. (1987) EMBOJ 6:390′-3907). The β-glucuronidase activity can be determined in plantaby means of a chromogenic substrate such as5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid in an activity stain(Jefferson et al. (1987) Plant Mol Biol Rep 5:387-405). To study thetissue specificity, the plant tissue is disected, embedded, stained andanalyzed as described (for example Bäumlein H et al. (1991) Mol GenGenet 225:121-128).

MUG (methylumbelliferylglucuronide) is used as substrate for thequantitative determination of the β-glucuronidase activity; it iscleaved into MU (methylumbelliferone) and glucuronic acid. Underalkaline conditions, this cleavage can be monitored quantitatively byfluorometry (excitation at 365 nm, measurement of the emission at 455nm; SpectroFluorimeter Thermo Life Sciences Fluoroscan) as described(Bustos M M et al. (1989) Plant Gell 1:839-853).

Example 4 Cloning the Promoters

To isolate the complete promoters of Seq ID NO: 1 or 2, genomic DNA isextracted from Arabidopsis thaliana (ectotype Landsberg erecta) asdescribed (Galbiati M et al. Funct Integr Genomics 2000, 20 1:25-34).The isolated DNA is employed as template DNA in a PCR, using thefollowing primers: Promoter Forward primer Reverse primer 761 (SEQ IDNO: 2) SEQ ID NO: 5 (76s) SEQ ID NO: 7 (76asl) 841 (SEQ ID NO: 3) SEQ IDNO: 8 (84s) SEQ ID NO: 10 (84asl) 76s (SEQ ID NO: 4) SEQ ID NO: 5 (76s)SEQ ID NO: 6 (76ass) 84s (SEQ ID NO: 5) SEQ ID NO: 8 (84s) SEQ ID NO: 9(84ass)

The amplification is carried out as follows:

-   -   80 ng genomic DNA    -   1× Expand™ Long Template PCR buffer    -   2.5 mM MgCl2,    -   350 μM of each dATP, dCTP, dGTP and dTTP    -   300 nM of each primer—(SEQ ID NO: 5 and 7 for promoter 761 and        SEQ ID NO 8 and 10 for promoter 84s)    -   2.5 units Expand™ Long Template polymerase (Roche Diagnostics).    -   in a final volume of 25 μl

The following temperature program is used (PTC-100TM model QfiV; MJResearch, Inc., Watertown, Massachussetts):

-   1 cycle with 120 seconds at 94° C.-   35 cycles with 10 seconds at 94° C., 30 seconds at 55° C. and 3    minutes at 68° C.-   1 cycle for 30 minutes 45 at 68° C.

The PCR products were cleaved with the restriction endonucleases SmaIand SalI and cloned into the vector pSUN::GUS. The resulting constructsare pSUN3-76L::GUS (FIG. 9), pSUN3-76S::GUS (FIG. 10), pSUN3-84L::GUS(FIG. 11) and pSUN3-84S::GUS (FIG. 11). After these constructs have beenstably transformed into Arabidopsis thaliana, RNA can be obtained fromthe various tissues, and the expression of the GUS gene can be shownqualitatively by RT-PCR and quantitatively by means of “Real Time” PCR.

The method for the quantitative “Real Time” PCR is described for examplein Bustin S A (2000) J Mol Endocrinol 25(2):169-93.

The Primers GUS for 5′-cac ttt tcc cgg caa taa cat-3′ GUS rev 5′-atc aggaag tga tgg agc atc-3′

were used for detecting and quantifying the GUS mRNA. The values werestandardized with the constitutively expressed tubulin. To detect andquantify tubulin, the primers TUB for 5′-gac cct gtc cca cct cca a-3′TUB rev 5′-tga gaa ctg cga ttg ttt gca-3′were used.

Example 5 TAIL-PCR

The “TAIL-PCR” is carried out in accordance with an adapted protocol ofthe method of Liu et al. (1995) Plant J 8(3):457-463 and Tsugeki et al.(1996) Plant J 10(3):479-489 (cf. FIG. 9). The following mastermix(quantities per reaction mix) is employed for a first PCR reaction:

-   -   11 μl steril H₂O (double-distilled)    -   2 μl primer stock solution of the specific primer 1 (5 mM)    -   3 μl AD2 primer stock solution (20 mM)    -   2 μl 10×PCR buffer    -   21 μl 10×dNTP    -   0.2 μl Taq polymerase

In a PCR vessel, 19 μl of this mastermix are pipetted to 1 μl of apreparation of genomic DNA of the target organism in question(preparation as described by Galbiati M et al. (2000) Funct IntegrGenomics 20(1):25-34)) and mixed thoroughly by pipetting.

The primary PCR reaction is carried out under the following conditions:

-   -   94° C. for 1 minute    -   four cycles with 94° C. for 10 seconds, 62° C. for 1 minute and        72° C. for 150 seconds    -   94° C. for 10 seconds, 25° C. for 3 minutes, 0.2° C./s to 72° C.        and 72° C. for 150 seconds    -   fourteen cycles at 94° C. for 10 seconds, 69° C. for 1 minute,        72° C. for 150 seconds, 94° C. for 10 seconds, 68° C. for 1        minute, 72° C. for 150 seconds, 94° C. for 10 seconds, 44° C.        for 1 minute and 72° C. for 150 seconds    -   72° C. for 5 minutes, then 4° C. until further use.

The product of the PCR reaction is diluted 1:50, and 1 μl of eachdiluted sample is used for a second PCR reaction (secondary PCR). Thefollowing mastermix is employed for this purpose (quantities perreaction mix):

-   -   12 μl of sterile H₂O (double distilled)    -   2 μl 10×PCR buffer (1.5 mM MgCl₂)    -   2 μl 10×dNTP    -   2 μl primer stock solution of the specific primer 2 (5 mM)    -   2 μl AD2 primer stock solution    -   0.2 μl Taq polymerase

In each case 20.2 μl of the second mastermix are added to in each case 1μl of the 1:50 diluted primary PCR product, and the secondary PCR iscarried out under the following conditions:

-   -   11 cycles at 94° C. for 10 seconds, 64° C. for 1 minute, 72° C.        for 150 seconds, 94° C. for 10 seconds, 64° C. for 1 minute,        72° C. for 150 seconds, 94° C. for 10 seconds, 44° C. for 1        minute, 72° C. for 150 seconds,    -   72° C. for 5 minutes, then 4° C. until further use.

The product of the PCR reaction is diluted 1:10, and 1 μl of eachdiluted sample is used for a third PCR reaction (tertiary PCR). Thefollowing mastermix is employed for this purpose (quantities perreaction mix):

-   -   18 μl of sterile H₂O (double distilled)    -   3 μl 10×PCR buffer (1.5 mM MgCl₂)    -   3 μl 10×dNTP    -   3 μl primer stock solution of the specific primer 3 (5 mM)    -   3 μl AD2 primer stock solution    -   0.5 μl Taq polymerase

In each case 30.3 μl of this mastermix are added to in each case

1 μl of the 1:10 diluted secondary PCR product, and the tertiary PCR iscarried out under the following conditions:

-   -   19 cycles at 94° C. for 15 seconds, 44° C. for 1 minute, 72° C.        for 150 seconds,    -   72° C. for 5 minutes, then 4° C. until further use.

In each case 5 μl of the products of the PCR 1, 2 and 3 of each sampleare separated on a 2% strength agarose gel. Those PCR products which,owing to the treated specific primers, show the expected size decrementare, if necessary, purified from the gel, reamplified with the primerpair which was used last, and then sequenced.

Reagents:

Taq polymerase 5U/μl

10×PCR buffer (1.5 mM MgCl₂)

10×dNTP stock solution: 2 mM

Primers:

Degenerate random primer (stock solutions 20 μM): AD1:5′-NTCGA(G/C)T(A/T)T(G/C)G(A/T)GTT-3′ AD2:5′-NGTCGA(G/C)(A/T)GANA(A/T)GAA-3′ AD5: 5′-(A/T)CAGNTG(A/T)TNGTNCTG-3′

Example 6 Inverse PCR (iPCR) for the Amplification of Insert-FlankingDNA

The “iPCR” is carried out in accordance with an adapted protocol of themethod of Long et al. (1993) PNAS 90:10370 (cf. FIG. 8):

-   -   1. Restriction of approx. 21 g of genomic DNA with BstYI for        approximately 2 hours at 37° C. in a total volume of 50 μl.    -   2. Ligation of 25 μl of the restriction mix with 3U T4-DNA        ligase at 15° C. overnight in a total volume of 300 μl.    -   3. Phenol/chloroform extraction and subsequent chloroform        extraction of the ligation mix. After ethanol precipitation,        take up DNA in 10 μl of sterile H₂O (double-distilled).    -   4. Employ 2.5 μl of the DNA solution for the PCR        -   Reaction mix:        -   2.5 μl of the DNA solution        -   10 μl 10×PCR buffer        -   2 μl dNTP (mixture of 10 mM each)        -   5 μl primer 1 (25 pmol)    -   5 μl primer 2 (25 pmol)    -   1,5 μl Taq polymerase    -   74 μl H₂O (double-distilled, sterile) to a total volume of 100        μl    -   PCR protocol: 4 minutes at 94° C. Then 35 cycles with 1 minute        at 94° C., 2 minutes at 55° C. and 3 minutes at 72° C. Finally,        8 minutes at 72° C., then 4° C. until further use.

The PCR product is checked by gel electrophoresis, purified andsubsequently sequenced as PCR product.

Example 6 Production of Transgenic Tagetes Plants

Tagetes seeds are sterilized and placed on germination medium (MSmedium; Murashige & Skoog (1962) Physiol Plant 15:473-497; pH 5.8, 2%sucrose). Germination takes place in a temperature/light/time intervalof 18 to 28° C./20 to 200 μE/3 to 16 weeks, but preferably at 21° C., 20to 70 μE, for 4 to 8 weeks.

All leaves of the plants which have developed in vitro by then areharvested and cut transverse to the central vein. The leaf explantsresulting therefrom, with a size of 10 to 60 mm², are stored during thepreparation in liquid MS medium at room temperature for not more than 2h.

Any Agrobacterium tumefaciens strain, but preferably a supervirulentstrain such as, for example, EHA105 with an appropriate binary plasmid,which may harbor a selection marker gene (preferably bar or pat) and oneor more trait or reporter genes (for example pS5KETO2 and pS5AP3PKETO2),is cultivated overnight and used for cocultivation with the leafmaterial. The bacterial strain can be cultured as follows: a singlecolony of the appropriate strain is inoculated in YEB (0.1% yeastextract, 0.5% beef extract, 0.5% peptone, 0.5% sucrose, 0.5% magnesiumsulfate×7H₂O) with 25 mg/l kanamycin and cultured at 28° C. for 16 to 20h. The bacterial suspension is then harvested by centrifugation at 6000g for 10 min, and resuspended in liquid MS medium so as to result in anOD₆₀₀ of about 0.1 to 0.8. This suspension is used for thecocultivaation together with the leaf material.

Immediately before the cocultivation, the MS medium in which the leaveshave been stored is replaced by the bacterial suspension. Incubation ofthe leaves in the agrobacterial suspension took place at roomtemperature with gentle shaking for 30 min. The infected explants arethen put on an MS medium solidified with agar (e.g. 0.8% plant agar(Duchefa, NL)), with growth regulators such as, for example, 3 mg/lbenzylaminopurine (BAP) and 1 mg/l indolylacetic acid (IAA). Theorientation of the leaves on the medium is immaterial. Cultivation ofthe explants takes place for 1 to 8 days, but preferably for 6 days,during which the following conditions can be used: light intensity: 30to 80 μmol/m²×sec, temperature: 22 to 24° C., 16/18-hour photoperiod.The cocultivated explants are then transferred to fresh MS medium,preferably with the same growth regulators, this second mediumadditionally containing an antibiotic to suppress bacterial growth.Timentin in a concentration of 200 to 500 mg/l is very suitable for thispurpose. The second selective component employed is one for selectingfor successful transformation. Phosphinothricin in a concentration of 1to 5 mg/l selects very efficiently, but other selective componentsaccording to the method to be used are also conceivable.

After one to three weeks in each case, the explants are transferred tofresh medium until plumules and small shoots develop, which are thentransferred to the same basal medium including timentin and PPT oralternative components with growth regulators, namely, for example, 0.5mg/l indolylbutyric acid (IBA) and 0.5 mg/l gibberellic acid GA3, forrooting. Rooted shoots can be transferred into the glasshouse.

In addition to the method described, the following advantageousmodifications are possible:

-   -   Before the explants are infected with the bacteria, they can be        preincubated on the medium described above for the cocultivation        for 1 to 12 days, preferably 3 to 4. This is followed by        infection, cocultivation and selective regeneration as described        above.    -   The pH for the regeneration (normally 5.8) can be lowered to pH        5.2. This improves control of the growth of agrobacteria.    -   Addition of AgNO₃ (3 to 10 mg/l) to the regeneration medium        improves the condition of the culture, including the        regeneration itself.    -   Components which reduce phenol formation and are known to the        skilled worker, such as, for example citric acid, ascorbic acid,        PVP and many others, have beneficial effects on the culture.    -   Liquid culture medium can also be used for the whole method. The        culture can also be incubated on commercially available supports        which are positioned on the liquid medium.

1. A method for the targeted transgenic expression of nucleic acidsequences in nonreproductive floral tissues of plants, comprising thefollowing steps, I. introduction of a transgenic expression cassetteinto plant cells, wherein the transgenic expression cassette comprisesat least the following elements a) at least one promoter sequenceselected from the group of sequences consisting of i.) the promotersequences of SEQ ID NO: 1 or 2 and ii.) functional equivalents of thepromoter sequences of SEQ ID NO: 1 or 2 with essentially the samepromoter activity as a promoter of SEQ ID NO: 1 or 2 and iii.)functional equivalent fragments of the sequences of i) or ii) withessentially the same promoter activity as a promoter of SEQ ID NO: 1 or2, and b) at least one further nucleic acid sequence, and c) optionallyfurther genetic control elements, wherein at least one promoter sequenceand one further nucleic acid sequence are functionally linked together,and the further nucleic acid sequence is heterologous in relation to thepromoter sequence, and II. selection of transgenic cells which comprisesaid expression cassette stably integrated into the genome, and III.regeneration of complete plants from said transgenic cells, wherein atleast one of the further nucleic acid sequences is expressed essentiallyin all nonreproductive floral tissues, but essentially not in the pollenand the ovaries.
 2. The method according to claim 1, wherein thefunctionally equivalent fragment comprises a sequence as shown in SEQ IDNO: 3 or
 4. 3. A method for identifying and/or isolating promoters ofgenes which encode a promoter having specificity for nonreproductivefloral tissue, wherein at least one nucleic acid sequence or a partthereof is employed in the identification and/or isolation, wherein saidnucleic acid sequence encodes an amino acid sequence which comprises atleast one sequence of SEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 or32 or a variation of these sequences.
 4. The method according to claim3, wherein said nucleic acid sequence comprises a sequence of SEQ ID NO:11, 13, 15, 17, 19 or
 21. 5. The method according to claim 3, whereinthe method is carried out with use of the polymerase chain reaction, andsaid nucleic acid sequence or a part thereof is employed as primer.
 6. Amethod for producing a transgenic expression cassette having specificityfor nonreproductive floral tissue, comprising the following steps: I.isolation of a promoter with specificity for nonreproductive floraltissue, where at least one nucleic acid sequence or a part thereof isemployed in the isolation, where said nucleic acid sequence encodes anamino acid sequence which comprises at least one sequence as shown inSEQ ID NO: 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 or a variation ofthese sequences, and II. functional linkage of said promoter with afurther nucleic acid sequence, where said nucleic acid sequence isheterologous in relation to the promoter.
 7. The method according toclaim 6, where said nucleic acid sequence comprises a sequence as shownin SEQ ID NO: 11, 13, 15, 17, 19 or
 21. 8. The method according to claim6, where the method is carried out with use of the polymerase chainreaction, and said nucleic acid sequence or a part thereof is employedas primer.
 9. A polypeptide comprising an amino acid sequence of SEQ IDNO: 16, 18, 20 or
 22. 10. A nucleic acid sequence encoding a polypeptideaccording to claim
 9. 11. The nucleic acid sequence according to claim10, comprising a sequence selected from the group of sequences of SEQ IDNO: 15, 17, 19 or 21 and the sequences derived therefrom as the resultof the degeneracy of the genetic code.
 12. (canceled)
 13. (canceled) 14.A transgenic expression cassette for the targeted transgenic expressionof nucleic acid sequences in nonreproductive floral tissues of plants,comprising a) at least one promoter sequence selected from the group ofsequences consisting of i) the promoter sequences of SEQ ID NO: 1 or 2and ii) functional equivalents of the promoter sequences of SEQ ID NO: 1or 2 with essentially the same promoter activity as a promoter of SEQ IDNO: 1 or 2 and iii) functionally equivalent fragments of the sequencesof i) or ii) with essentially the same promoter activity as a promoterof SEQ ID NO: 1 or 2, and b) at least one further nucleic acid sequence,and c) optionally further genetic control elements, where at least onepromoter sequence and one further nucleic acid sequence are functionallylinked together, and the further nucleic acid sequence is heterologousin relation to the promoter sequence.
 15. The transgenic expressioncassette according to claim 14, wherein the functionally equivalentfragment comprises a sequence of SEQ ID NO: 3 or
 4. 16. The transgenicexpression cassette according to claim 14, where a) the nucleic acidsequence to be expressed is functionally linked with further geneticcontrol sequences, or b) the expression cassette comprises additionallyfunctional elements, or c) a) and b) apply.
 17. The transgenicexpression cassette according to claim 14, wherein the nucleic acidsequence to be expressed transgenically makes possible a) the expressionof a protein encoded by said nucleic acid sequence, or b) the expressionof a sense-RNA, anti-sense RNA or double-stranded RNA encoded by saidnucleic acid sequence.
 18. The transgenic expression cassette accordingto claim 14, wherein the nucleic acid sequence to be expressedtransgenically is selected from the group of nucleic acid sequencesencoding chalcone synthases, phenyalanine ammonium lyases, photolyases,deoxyxylulose-5-phosphate synthases, phytoene synthases, phytoenedesaturases, lycopene cyclases, hydroxylases, “antifreeze” polypeptides,CBF1-transcription activators, glutamate dehydrogenases,calcium-dependent protein kinases, calcineurin, farnesyltransferases,ferritin, oxalate oxidases, DREB1A factor, trehalose-phosphatephosphatases, chitinases, glucanases, ribosome-inactivating protein,lysozyme, Bacillus thuringiensis endotoxins, amylase inhibitors,protease inhibitors, lectins, RNAses, ribozymes, endochitinase,cytochrome P-450, acetyl-CoA carboxylases, amino acid transporters,monosaccharide-transporters, lycopine cyklases, carotene ketolases,endoxyloglucan transferases, Δ6-acyllipid desaturases, Δ6-desaturases,Δ5-fatty acid desaturases, Δ6-elongases and IPP-isomerases.
 19. Thetransgenic expression cassette according to claim 14, wherein thenucleic acid sequence to be expressed transgenically is selected fromthe group of nucleic acid sequences described by GenBank Acc.-No.:M20308, BAB00748, U62549, U77378, S78423, U32624, L25042, X92657,AJ002399, D45881, AF163819, AB044391, AJ222980 and AF078796.
 20. Atransgenic expression vector comprising an expression cassette accordingto claim
 14. 21. A transgenic organism, transformed with an expressioncassette of claim
 14. 22. The transgenic organism according to claim 21selected from the group consisting of bacteria, yeasts, fungi, non-humananimal and plant organisms or of cells, cell cultures, parts, tissues,organs or propagation material derived therefrom.
 23. The transgenicorganism as claimed in claim 21 selected from the group of agriculturalcrop plants.
 24. A method for producing human or animal foods, seeds,pharmaceuticals or fine chemicals comprising culturing or growing thetransgenic organism according to claim 21 or cells, cell cultures,parts, tissues, organs or propagation material derived therefrom.
 25. Amethod for producing pharmaceuticals or fine chemicals in transgenicorganisms according to claim 21 or cells, cell cultures, parts, tissues,organs or propagation material derived therefrom, where the transgenicorganism or cells, cell cultures, parts, tissues, organs or propagationmaterial derived from them is/are cultured or grown, and the desiredpharmaceutical or the desired fine chemical is isolated.